Plasmid PXL3179 or NV1FGF

ABSTRACT

A prokaryotic recombinant host cell comprising a heterologous replication initiation protein that activates a conditional origin of replication and an extrachromosomal DNA molecule comprising a heterologous therapeutic gene and a conditional origin of replication whose functionality in the prokaryotic recombinant host cell requires a replication initiating protein which is foreign to the host cell is described. The host cell may comprise a pir gene having at least one mutation, which may occur in the pir gene copy number control region, the pir gene leucine zipper-like motif, or the pir gene DNA binding region.

This application is a continuation of application Ser. No. 11/978,614,filed Oct. 30, 2007, which is a continuation of application Ser. No.10/684,830, filed Oct. 15, 2003, now U.S. Pat. No. 7,364,894, all ofwhich are incorporated by reference in their entirety.

The present invention relates to a novel conditional replication DNAmolecule which can be used in gene therapy or for the production ofrecombinant proteins. The novel DNA molecules according to the presentinvention are designated pCOR™ herein after.

Gene therapy consists in correcting a deficiency or an anomaly byintroducing genetic information into the affected organ or cell. Thisinformation may be introduced either in vitro into a cell extracted fromthe organ and then reinjected into the body, or in vivo, directly intothe target tissue. As a molecule of high molecular weight and ofnegative charge, DNA has difficulty in spontaneously crossingphospholipid cell membranes. Various vectors are thus used in order toenable gene transfer to take place: viral vectors, on the one hand, andnatural or synthetic chemical and/or biochemical vectors, on the otherhand.

Viral vectors (retroviruses, adenoviruses, adeno-associated viruses,etc.) are very effective, in particular for crossing membranes, butpresent a certain number of risks such as pathogenicity, recombination,replication, and immunogenicity.

Chemical and/or biochemical vectors allow these risks to be avoided (forreviews, see Behr, 1993, Cotten and Wagner 1993). These are, forexample, cations (calcium phosphate, DEAE-dextran, etc.) which act byforming precipitates with DNA, which may be “phagocytosed” by the cells.They may also be liposomes in which the DNA is incorporated and whichfuse with the plasma membrane. Synthetic gene-transfer vectors aregenerally lipids or cationic polymers which complex the DNA and formwith it a particle bearing positive surface charges. As illustrations ofvectors of this type, mention may be made in particular ofdioctadecylamidoglycylspermine (DOGS, Transfectam™) orN-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammoniun (DOTMA,Lipofectin™).

However, the use of chemical and/or biochemical vectors or naked DNAimplies the possibility of producing large amounts of DNA ofpharmacological purity. The reason for this is that in gene therapytechniques, the medicinal product consists of the DNA itself and it isessential to be able to manufacture, in suitable amounts, DNAs havingproperties which are appropriate for therapeutic use in man.

In the case of non-viral vectorology, the vectors used are plasmids ofbacterial origin. The plasmids generally used in gene therapy carry (i)an origin of replication, (ii) a marker gene such as a gene forresistance to an antibiotic (kanamycin, ampicillin, etc.) and (iii) oneor more transgenes with sequences necessary for their expression(enhancer(s), promoter(s), polyadenylation sequences, etc.). However,the technology currently available is not entirely satisfactory.

On the one hand, the risk remains of dissemination in the body. Thus, abacterium which is present in the body can, at low frequency, receivethis plasmid. There is a greater likelihood of this taking place if itinvolves an in vivo gene therapy treatment in which the DNA may bedisseminated in the body of the patient and may come into contact withbacteria which infect this patient or bacteria of the commensal flora.If the bacterium receiving the plasmid is an enterobacterium, such as E.coli, this plasmid can be replicated. Such an event then leads todissemination of the therapeutic gene. Insofar as the therapeutic genesused in gene therapy treatments can code, for example, for a lymphokine,a growth factor, an anti-oncogene or a protein whose function isdefective in the host and which thus makes it possible to correct agenetic defect, the dissemination of some of these genes could haveunforeseeable and worrying effects (for example if a pathogenicbacterium acquired a human growth factor gene).

On the other hand, the plasmids generally used in non-viral gene therapyalso possess a marker for resistance to an antibiotic (ampicillin,kanamycin, etc.). The bacterium acquiring such a plasmid thus has anundeniable selective advantage since any antibiotic therapy, using anantibiotic from the same family as that which selects the plasmidresistance gene, will lead to selection of the plasmid in question. Inthis respect, ampicillin belongs to the α-lactams, which is the familyof antibiotics which is most frequently used worldwide. The use inbacteria of selection markers which are not antibiotic-resistance geneswould thus be particularly advantageous. This would avoid the selectionof bacteria which may have received a plasmid carrying such a marker.

It is thus particularly important to seek to limit the dissemination oftherapeutic genes and resistance genes as much as possible.

The subject of the present invention is specifically to propose novelDNA molecules which can be used in gene therapy or for the production ofrecombinant proteins in vitro and which replicate only in cells whichcan complement certain functions of these non-viral vectors.

The invention also relates to a particularly effective method forpreparing these DNA molecules.

The DNA molecules claimed have the advantage of removing the risksassociated with dissemination of the plasmid, such as (1) replicationand dissemination, which can lead to uncontrolled overexpression of thetherapeutic gene, (2) dissemination and expression of resistance genes.The genetic information contained in the DNA molecules according to theinvention effectively comprises the therapeutic gene(s) and the signalsfor regulating its (their) expression, a functional conditional originof replication which greatly limits the host cell spectrum of thisplasmid, a selection marker of reduced size which is preferablydifferent from a gene which imparts resistance to an antibiotic and,where appropriate, a DNA fragment which allows the resolution of plasmidmultimers. The probability of these molecules (and thus the geneticinformation which they contain) being transferred to a microorganism,and maintained stably, is very limited.

Lastly, the vectors according to the invention, also referred to asminiplasmids on account of their circular structure, their reduced sizeand their supercoiled form, have the following additional advantages: onaccount of their size which is reduced in comparison with theColE1-derived plasmids used conventionally, the DNA molecules accordingto the invention potentially have better in vivo bioavailability, andthe DNA molecules or pCOR stay in a stable extrachromosomal form in thehost prokaryotic or eukaryotic cells that do not contain the initiatingprotein. In particular, they have improved capacities of cellpenetration and distribution. Thus, it is acknowledged that thediffusion coefficient in tissues is inversely proportional to themolecular weight (Jain, 1987). Similarly, in the cell, high molecularweight molecules have poorer permeability across the plasma membrane. Inaddition, in order for the plasmid to pass into the nucleus, which isessential for its expression, the high molecular weight is also adisadvantage, the nuclear pores imposing a size limit for diffusion intothe nucleus (Landford et al., 1986). The reduction in size of thenon-therapeutic parts of the DNA molecule (origin of replication andselection gene in particular) according to the invention also makes itpossible to decrease the size of the DNA molecules. The part whichallows the replication and selection of this plasmid in the bacterium (1kb) is decreased by a factor of 3, counting, for example, 3 kb for theorigin of replication and the resistance marker vector part. Thisdecrease (i) in molecular weight and (ii) in negative charge impartsimproved tissue, cellular and nuclear bioavailability and diffusion tothe molecules of the invention.

More precisely, the present invention relates to a circular DNAmolecule, which is useful in gene therapy, this molecule comprising atleast one nucleic acid sequence of interest, characterized in that theregion which allows its replication comprises an origin of replicationwhose functionality in a host cell requires the presence of at least onespecific protein which is foreign to the said host cell.

This DNA molecule may be in single- or double-stranded form andadvantageously possesses a supercoiled form.

For the purposes of the present invention, the host cells used can be ofvarious origins. They can be eukaryotic or prokaryotic cells. Accordingto a preferred embodiment of the invention, they are prokaryotic cells.

The replication of bacterial plasmids conventionally requires thepresence of at least one protein, which is coded for by the host cell,of the RNA polymerase, Rnase, DNA polymerase, etc. type. For the reasonsalready explained above, it is not possible to overcome entirely, withthis type of replication, any possible risks of dissemination in thetreated organism. Advantageously, the functionality of the origin ofreplication of the DNA molecule according to the invention requires thepresence of a specific protein which is foreign to the host cell. Thesignificance of this characteristic is to reduce the host spectrum ofthe claimed plasmid to specific strains that express this initiatorprotein. The DNA molecule developed within the context of the presentinvention thus advantageously possesses a so-called conditional originof replication.

The conditional origin of replication used according to the presentinvention may originate from plasmids or bacteriophages which share thefollowing characteristics: they contain in their origin of replicationrepeat sequences, or iterons, and they code for at least onereplication-initiating protein (Rep) which is specific to them. By wayof example, mention may be made of the conditional replication systemsof the following plasmids and bacteriophages:

specific initiator plasmid or bacteriophage protein RK2 (Stalker et al.,1981) TrfA R1 (Ryder et al., 1981) RepA pSC101 (Vocke and Bastia, 1983)RepA F (Murotsu et al., 1981) protein E Rts1 (Itoh et al., 1982, 1987)RepA RSF1010 (Miao et al., 1995) RepC P1 (Abeles et al., 1984) RepA P4(Flensburg and Calendar, 1987) alpha protein lambda (Moore et al., 1981)protein O phi 82 (Moore et al., 1981) protein O from phi 82 phi 80protein O from phi 80

According to a preferred embodiment of the invention, the origin ofreplication used in the DNA molecules claimed is derived from a naturalE. coli plasmid referred to as R6K.

The replication functions of R6K are grouped together in a 5.5 kbp DNAfragment (FIG. 1) comprising 3 origins of replication α, β, and γ (γ andα providing 90% of the replication) and an operon coding for the Hreplication-initiator protein and the protein Bis. The minimum amount ofgenetic information required to maintain this plasmid at itscharacteristic number of copies (15 copies per genome) is contained intwo elements: the 400 bp of ori γ and the gene pir, whose product is theΠ initiator protein.

Ori γ may be divided into two functional parts: the core part and theactivator element (FIG. 1). The core part, which is essential forreplication, contains the iterons (7 direct repeats of 22 bp) to whichthe Π protein represented in SEQ ID No. 1 becomes bound, and flankingsegments, which are targets of the host proteins (IHF, DnaA).

According to a preferred mode of the invention, the origin ofreplication of the vector claimed consists entirely or partially of thisγ origin of replication of the plasmid R6K and more preferably, entirelyor partially of SEQ ID No. 1 or one of its derivatives.

The origin of replication described above, which has the advantage ofbeing of very limited size, is functional solely in the presence of aspecific initiator protein, protein Pi, produced by the gene pir (SEQ IDNo. 2). Since this protein can act in trans, it is possible tophysically dissociate the ori gamma from the pir gene, which may beintroduced into the genome of the cell which is chosen as the specifichost for these plasmids. Mutations in Π may alter its inhibitoryfunctions (Inuzuka and Wada, 1985) and lead to an increase in the numberof copies of the R6K derivatives, up to more than 10 times the initialnumber of copies. These substitutions may be within a domain of 40 aminoacids, which therefore appears to be responsible for the control by Π ofthe number of plasmid copies (FIG. 2), or in other regions of the Πprotein.

According to an advantageous embodiment of the present invention, the Πprotein, expressed in the host cell, results from the expression of thegene represented in SEQ ID No. 2 or one of its derivatives as definedabove and more particularly of the gene pir 116 which comprises amutation when compared with the pir gene. This mutation corresponds tothe replacement of a proline by a leucine at position 106 from the startcodon. In this context, the number of copies of the R6K derivatives isabout 250 copies per genome.

For the purposes of the present invention, the term derivative denotesany sequence which differs from the sequence considered, obtained by oneor more modifications of genetic and/or chemical nature, as well as anysequence which hybridizes with these sequences or fragments thereof andwhose product possesses the activity indicated with regard to thereplication-initiator protein Π. The term modification of the geneticand/or chemical nature may be understood to refer to any mutation,substitution, deletion, addition and/or modification of one or moreresidues. The term derivative also comprises the sequences homologouswith the sequence considered, derived from other cellular sources and inparticular cells of human origin, or from other organisms, andpossessing an activity of the same type. Such homologous sequences maybe obtained by hybridization experiments. The hybridizations may beperformed starting with nucleic acid libraries, using the nativesequence or a fragment thereof as probe, under conventional conditionsof stringency (Maniatis et al., cf. General techniques of molecularbiology), or, preferably, under conditions of high stringency.

Besides a conditional origin of replication as defined above, the DNAmolecules claimed contain a region comprising one (or more) gene(s)which make it possible to ensure selection of the DNA molecule in thechosen host.

This may be a conventional marker of gene type which imparts resistanceto an antibiotic, such as kanamycin, ampicillin, chloramphenicol,streptomycin, spectinomycin, lividomycin or the like.

However, according to a preferred embodiment of the invention, thisregion is different from a gene which imparts resistance to anantibiotic. It may thus be a gene whose product is essential for theviability of the host envisaged, under defined culturing conditions. Itmay be, for example:

a gene coding for a suppressor tRNA, of natural or synthetic origin.This is, more preferably, an amber codon tRNA (TAG)

a gene whose product is necessary for metabolism of the cell, undercertain culturing conditions, namely a gene involved in the biosynthesisof a metabolite (amino acid, vitamin, etc.), or a catabolism gene whichmakes it possible to assimilate a substance present in the culturemedium (specific nitrogen or carbon source), etc.

According to a preferred mode of the invention, this region contains anexpression cassette of a gene coding for a suppressor tRNA for specificcodons. This latter may be chosen, in particular, from those coding forphenylalanine, cysteine, proline, alanine and histidine amino acids. Itis more particularly a suppressor tRNA for amber codons (TAG).

In this particular case, the system used to select, in the host cells,the DNA molecules which are the subject of the present inventionincludes two elements: 1) on the DNA molecule, a gene coding for asuppressor transfer RNA for the amber codon (TAG) which constitutes theselection marker, known as (sup) gene and 2) a specific host, one ofwhose genes, which is essential under certain culture conditions,contains an amber TAG codon. This cell may grow, under the cultureconditions for which the product of the gene containing the TAG codon isessential, only if the plasmid allowing the expression of sup is presentin the cell. The culture conditions thus constitute the pressure forselection of the DNA molecule. The sup genes used may be of naturalorigin (Glass et al., 1982) or may originate from a syntheticconstruction (Normanly et al., 1986, Kleina et al., 1990).

Such a system offers great flexibility insofar as, depending on the genecontaining an amber mutation, it is possible to determine variousselective media. In the bacterium Lactococcus lactis for example, theamber codon is located in a purine biosynthesis gene. This allows theselection of the plasmid carrying the gene coding for the suppressortRNA when the bacteria multiply in milk. Such a marker has the advantageof being very small and of containing no “foreign” sequences,originating from phages or transposons.

According to a particular embodiment of the invention, the DNA moleculealso comprises a DNA fragment, the target for site-specificrecombinases, which allows the resolution of plasmid multimers.

Thus, such a fragment, introduced on to a DNA molecule which is circularand whose origin of replication is, for example, ori gamma, allows theresolution of multimers of such a plasmid. Such multimers are observed,in particular, when the DNA molecule is prepared in a strain carrying amutated allele of pir, such as pir116, which makes it possible toincrease the number of copies of the R6K derivatives.

This recombination may be achieved by means of various systems whichentail site-specific recombination between sequences. More preferably,the site-specific recombination of the invention is obtained by means ofspecific intramolecular recombination sequences which are capable ofrecombining with each other in the presence of specific proteins,generally referred to as recombinases. In this specific case, these arethe recombinases XerC and XerD. For this reason, the DNA moleculesaccording to the invention generally also comprise a sequence whichallows this site-specific recombination. The specific recombinationsystem present in the genetic constructions according to the invention(recombinases and specific recognition site) may be of differentorigins. In particular, the specific sequences and the recombinases usedmay belong to different structural classes, and in particular to thetransposon Tn3 resolvase family or to the bacteriophage lambda integrasefamily. Among the recombinases belonging to the transposon Tn3 family,mention may be made in particular of the resolvase of transposon Tn3 orof transposons Tn21 and Tn522 (Stark et al., 1992); the Gin invertase ofbacteriophage mu or alternatively plasmid resolvases, such as that ofthe par fragment of RP4 (Abert et al., Mol. Microbiol. 12 (1994) 131).Among the recombinases belonging to the bacteriophage λ integrasefamily, mention may be made in particular of the integrase of the phageslambda (Landy et al., Science 197 (1977) 1147), P22 and Φ80 (Leong etal., J. Biol. Chem. 260 (1985) 4468), HP1 of Haemophilus influenzae(Hauser et al., J. Biol. Chem. 267 (1992) 6859), the Cre integrase ofphage P1, the integrase of plasmid pSAM2 (EP 350 341) or alternativelythe FLP recombinase of the 2 μl plasmid and the XerC and XerDrecombinases from E. coli.

Preferably, the DNA molecules which form the subject of the presentinvention contain the fragment cer from the natural E. coli plasmidColE1. The cer fragment used is a 382 bp HpaII fragment from ColE1 whichhas been shown to bring about, in cis, the resolution of plasmidmultimers (Summers et al., 1984; Leung et al., 1985). It is alsopossible to use a HpaII-TaqI fragment of smaller size (280 bp) or asmaller fragment (about 220 bp), contained in the HpaII fragment, whichfragments possess the same properties (Summers and Sherratt, 1988). Thisresolution takes place by way of a specific intramolecularrecombination, which involves four proteins encoded by the genome of E.coli: ArgR, PepA, XerC and XerD (Stirling et al., 1988, 1989; Colloms etal., 1990, Blakely et al., 1993). It was found that insertion of thefragment cer from the natural E. coli plasmid ColE1 allows to obtain ahigh resolution of plasmids multimers, thereby resulting in highproportion of monomers in a reproducible manner. This is particularlyunexpected as it has been shown that the insertion of the cer site intoa minicircle which contains the ColE1 origin of replication frompBluescript SK+ did not result in efficient multimer resolution (Kreisset al., Appl. Microbiol. Biotechnol, 49:560-567 (1998)), and thuseffective resolution in cis of plasmids is unpredictable and seems todepend on the plasmid conformation. In the case of the pCOR plasmid, aneffective cis resolution is reached when cer is present on the pCOR,thereby resulting in a unexpectedly high monomers of pCOR in areproducible manner.

In this respect, it is particularly advantageous to use all or part ofthe cer fragment of ColE1 or one of its derivatives as defined above.

According to an implementation variant, the DNA molecules of theinvention may also comprise a sequence capable of interactingspecifically with a ligand. Preferably, this is a sequence capable offorming, by hybridization, a triple helix with a specificoligonucleotide. This sequence thus makes it possible to purify themolecules of the invention by selective hybridization with acomplementary oligonucleotide immobilized on a support (see applicationWO 96/18744 and WO 02/07727). The sequence may be naturally present inthe origin of replication of the plasmid as described in US publicationapplication 2003/186268 of the Applicant, or naturally present in thetransgene as described in WO 02/07727, and alternatively can bepositioned at any site in the DNA molecule of the invention, providedthat it does not affect the functionality of the gene of interest and ofthe origin of replication. Formation of a triple helix by hybridizationthus occurs between the oligonucleotide and the specific complementarysequence present in the DNA. In this connection, to obtain the bestyields and the best selectivity, an oligonucleotide and a specificsequence which are fully complementary are used in the method of theinvention. These can be, in particular, an oligonucleotide poly(CTT) anda specific sequence poly(GAA). For example, oligonucleotides containingrepeated motifs such as CTT are capable of forming a triple helix with aspecific sequence containing complementary units (GAA). The sequence inquestion can, in particular, be a region containing 7, 14 or 17 GAAunits, and in the oligonucleotides a corresponding numbers of repeatCTT. In this case, the oligonucleotide binds in an antiparallelorientation to the polypurine strand. These triple helices are stableonly in the presence of Mg²⁺ (Vasquez et al., Biochemistry, 34:7243-7251 (1995); Beal and Dervan, Science, 251: 1360-1363 (1991)).

As stated above, the specific sequence can be a sequence naturallypresent in the pCOR, or may be a synthetic sequence introducedartificially in the latter. It is especially advantageous to use anoligonucleotide capable of forming a triple helix with a sequencenaturally present in the pCOR, for example in the origin of replicationof a plasmid or in a marker gene. The synthesis of oligonucleotidescapable of forming triple helices with these naturalhomopurine-homopyrimidine regions is particularly advantageous, as itmay be applied to unmodified pCOR plasmids. Particularly preferredtarget sequences which can form triplex structures with particularoligonucleotides have been identified in ColE1 and in pCOR origins ofreplication. ColE1-derived plasmids contain a 12-mer homopurine sequence(5′-AGAAAAAAAGGA-3′) (SEQ ID NO: 33) mapped upstream of the RNA-IItranscript involved in plasmid replication (Lacatena et al., Nature,294: 623 (1981)). This sequence forms a stable triplex structure withthe 12-mer complementary 5′-TCTTTTTTTCCT-3′ (SEQ ID NO: 34)oligonucleotide. The pCOR backbone contains a homopurine stretch of 14non repetitive bases (5′-AAGAAAAAAAAGAA-3′) (SEQ ID NO: 35) located inthe A+T-rich segment of the γ origin replicon of pCOR (Levchenko et al.,Nucleic Acids Res., 24:1936 (1996)). This sequence forms a stabletriplex structure with the 14-mer complementary oligonucleotide5′-TTCTTTTTTTTCTT-3′ (SEQ ID NO: 36). The corresponding oligonucleotides5′-TCTTTTTTTCCT-3′ (SEQ ID NO: 37) and 5′-TTCTTTTTTTTCTT-3′ (SEQ ID NO:38) efficiently and specifically target their respective complementarysequences located within the origin of replication of either ColE1 orior pCOR (oriγ). Also, use of an oligonucleotide capable of forming atriple helix with a sequence present in an origin of replication or amarker gene is especially advantageous, since it makes it possible, withthe same oligonucleotide, to purify any DNA containing the said originof replication or said marker gene. Hence it is not necessary to modifythe plasmid or the double-stranded DNA in order to incorporate anartificial specific sequence in it.

Although fully complementary sequences are preferred, it is understood,however, that some mismatches may be tolerated between the sequence ofthe oligonucleotide and the sequence present in the DNA, provided theydo not lead to too great a loss of affinity. The sequence5′-AAAAAAGGGAATAAGGG-3′ (SEQ ID NO: 39) present in the E. coliβ-lactamase gene may be mentioned. In this case, the thymineinterrupting the polypurine sequence may be recognized by a guanine ofthe third strand, thereby forming a G*TA triplet which it is stable whenflanked by two T*AT triplets (Kiessling et al., Biochemistry, 31:2829-2834 (1992)).

According to a particular embodiment, the oligonucleotides used maycomprise the sequence (CCT)_(n) (SEQ ID NO: 41), the sequence (CT)_(n)(SEQ ID NO: 42) or the sequence (CTT)_(n) (SEQ ID NO: 43), in which n isan integer between 1 and 15 inclusive. It is especially advantageous touse sequences of the type (CT)_(n) (SEQ ID NO: 42) or (CTT)_(n) (SEQ IDNO: 43). Oligonucleotides may also combine (CCT), (CT) or (CTT) units.

The oligonucleotides used may be natural (composed of unmodified naturalbases) or chemically modified. In particular, the oligonucleotide mayadvantageously possess certain chemical modifications enabling itsresistance to or its protection against nucleases, or its affinity forthe specific sequence, to be increased.

As a DNA molecule representative of the present invention, the plasmidpXL2774 and its derivatives may be claimed most particularly. For thepurposes of the invention, the term derivative is understood to refer toany construction derived from pXL2774 and containing one or more genesof interest other than the luciferase gene. Mention may also be made ofthe plasmids pXL3029, pXL3030, and plasmid pXL3179 or NV1FGF containingan expression cassette of a therapeutic gene. In a most preferredembodiment, the invention relates to a pCOR comprising the FGFa or FGF-1gene as described in U.S. Pat. No. 4,686,113 of the Applicant, which isdesignated pXL 3179 or NV1FGF.

The present invention also relates to the development of a process forthe construction of specific host cells, which are particularlyeffective for the production of these therapeutic DNA molecules.

Another subject of the present invention relates to a process for theproduction of a circular DNA molecule, characterized in that a host cellis cultured containing at least one DNA molecule as defined above and aprotein, which may or may not be expressed in situ, which conditions thefunctionality of the origin of replication of the said DNA molecule,which is specific and which is foreign to the said host cell, underconditions which allow the selection of host cells transformed by thesaid DNA molecules.

More preferably, the protein which conditions the functionality of theorigin of replication of the DNA molecule is expressed in situ from acorresponding gene. The gene coding for the replication-initiatingprotein may be carried by a subsidiary replicon, which is compatiblewith the derivatives of the conditional origin of replication used orwhich may be introduced into the genome of the host cell byrecombination, by means of a transposon, a bacteriophage or any othervector. In the particular case in which the gene expressing the proteinis placed on a subsidiary replicon, the latter also contains a promoterregion for functional transcription in the cell, as well as a regionwhich is located at the 3′ end and which specifies a transcriptiontermination signal. As regards the promoter region, this may be apromoter region which is naturally responsible for expressing the geneunder consideration when the latter is capable of functioning in thecell. It may also be a case of regions of different origin (responsiblefor expressing other proteins), or even of synthetic origin. Inparticular, it may be a case of promoter sequences for prokaryotic orbacteriophage genes. For example, it may be a case of promoter sequencesobtained from the cell genome.

As genes coding for the replication-initiating protein, use may be madeeither of wild-type genes or of mutated alleles which make it possibleto obtain an increased number of copies of the plasmids (or derivatives)specific for the initiator protein which conditions the functionality ofthe origin of replication used in the DNA molecule.

Such mutants have been described in particular for the plasmids R6K(Inuzuka and Wada, 1985; Greener et al., (1990), Rts1 (Terawaki andItoh, 1985, Terawaki et al., 1990; Zeng et al., 1990), F (Seelke et al.,1982; Helsberg et al., 1985; Kawasaki et al., 1991), RK2 (Durland etal., 1990; Haugan et al., 1992, 1995), pSC101 (Xia et al., 1991; Goebelet al., 1991; Fang et al., 1993).

In the particular case in which the DNA molecule used possesses anorigin of replication derived from the plasmid R6K, the initiatorprotein is a derivative of the H protein of this same plasmid. It isparticularly advantageous to express a mutated form of this proteinwhich is capable of increasing the number of initial copies appreciably.To do this, the gene incorporated into the host cell is preferablyrepresented by all or part of the sequence represented in SEQ ID No. 2or one of its derivatives and more preferably by the pir116 gene. Theassociated mutation corresponds to the replacement of a proline by aleucine. According to a particular embodiment of the invention, thispir116 gene is directly incorporated into the host cell genome.

Advantageously, one of the genes of the specific host cell, which isessential under the culture conditions chosen, contains a specific codonwhich is recognizable by the selected suppressor tRNA in the DNAmolecule. According to a preferred mode of the invention, this is anamber TAG codon. In this particular case, the cell may grow, underculture conditions for which the product of the gene containing the TAGcodon is essential, only if the plasmid allowing the expression of supis present in the host cell. The culture conditions thus constitute thepressure for selection of the DNA molecule.

Preferably, the gene containing the amber codon is a gene involved inthe biosynthesis of an amino acid, arginine. This gene, argE, codes foran N-acetylornithinase (Meinnel et al., 1992) and in this case containsa TAG codon corresponding to a point mutation Gln-53 (CAG)->TAG; thepressure for selection of the plasmid carrying the sup gene is thenprovided by culturing in minimal M9 medium (Maniatis et al., 1989).However, this could also be, for example, a gene for biosynthesis of avitamin or a nucleic acid base, or alternatively a gene which allows aspecific nitrogen or carbon source to be used or any other gene whosefunctionality is essential for cellular viability under the chosenculture conditions.

The host cell is preferably chosen from E. coli strains and is morepreferably represented by the strain E. coli XAC-J.

According to a specific embodiment of the invention, the host cell usedin the claimed process is a cell of the E. coli strain XAC-1, containingthe pir116 gene in its genome and transformed by the plasmid pXL2774 orone of its derivatives.

According to an advantageous variant of the invention, the host cellused in the process claimed is a prokaryotic cell in which the endA1gene or a homologous gene is inactivated. The endA gene codes forendonuclease I of E. coli. This periplasmic enzyme has a non-specificactivity of cleaving double-stranded DNA (Lehman, I. R., G. G. Roussosand E. A. Pratt (1962) J. Biol. Chem. 237: 819-828; Wright M. (1971) J.Bacteriol. 107: 87-94). A study carried out on various strains ofEscherichia coli (wild-type or endA) showed that the degradation ofplasmid DNA incubated in extracts of these bacterial strains existed inthe endA+ strains but not in the endA mutants. (Wnendt S. (1994)BioTechniques 17: 270-272). The quality of the plasmid DNA isolated fromendA+ strains or from endA mutants was studied by the company Promegausing their purification system (Shoenfeld, T., J. Mendez, D. Storts, E.Portman, B. †Patterson, J. Frederiksen and C. Smith. 1995. Effects ofbacterial strains carrying the endA1 genotype on DNA quality isolatedwith Wizard plasmid purification systems. Promega notes 53). Theirconclusion is as follows: the quality of the DNA prepared from endAmutants is, overall, better than that of DNA prepared in the endA+strains tested.

The quality of the plasmid DNA preparations is thus affected by anycontamination with this endonuclease (relatively long-term degradationof the DNA).

The deletion or mutation of the endA gene can be envisaged withoutdifficulty insofar as the mutants no longer having this endonucleaseactivity behave on the whole like wild-type bacteria (Dtirwald, H. andH. Hoffmann-Berling (1968) J. Mol. Biol. 34: 331-346).

The endA1 gene can be inactivated by mutation, total or partialdeletion, disruption, etc. Inactivation of the endA gene of the E. colistrain chosen to produce the pCOR plasmids can be achieved moreparticularly by transferring, by means of the PI bacteriophage, theΔendA::Tc^(R) deletion described by Cherepanov and Wackernagel(Cherepanov, P. P. and W. Wackernagel. 1995. Gene disruption inEscherichia coli: Tc^(R) and Km^(R) cassettes with the option ofFlp-catalyzed excision of the antibiotic-resistance determinant. Gene158:9-14) or by exchanging the wild-type allele present in the genome ofthe bacterium of interest with a mutated or deleted allele of endA, byhomologous recombination. The use of this type of strain in the contextof the present invention makes it possible advantageously to improve thequality of the DNA produced.

The invention also relates to any recombinant cell containing a DNAmolecule as defined above. This may be a cell of various origins, ofeukaryotic, prokaryotic, etc. type.

According to another embodiment of the invention, the E. coli XAC-1 hostcell used in the process claimed is designated TEX1, and comprises atraD gene, or a homologous gene thereof, inactivated to abolish F′transfer. The traD is at the 5′ end of one of the tra operon and encodesa 81.7 kDa membrane protein that is directly involved in DNA transferand DNA metabolism (Frost et al., Microbiology Reviews, 1994, 58:162-210). traD mutants do not transfer DNA (Panicker et al., J.Bacteriol., 1985, 162:584-590). The episomal traD gene may beinactivated by mutation, total or partial deletion, or disruption usingmethods well known to those of skill in the art (See Example 9). Onemethod of inactivating this gene is described in Example 1, and theresulting E. coli XAC-1 pir116 endA⁻ traD⁻ strain so obtained isdesignated TEX1 (Soubrier et al., Gene Therapy, 1999, 6: 1482-1488).

According to one embodiment of the invention, the host cell used in theclaimed process is a cell of the E. coli strain XAC-1, containing thepir116 mutation combined with the pir42 mutation. The pir116 and pir42mutations affect different domains of the pi protein. The pir116mutation affects the copy number control region, whereas the pir42mutation affects the putative leucine zipper motif, as displayed in FIG.11. The nucleotide and amino acid sequences of the pir gene containingthe pir116 and pir42 mutations are set forth in FIG. 12 and SEQ ID NOs:21 and 22, respectively. The pir42 mutation comprises a C to Ttransition at position 124 from the methionine initiator codon, and thusresults in substitution of the proline at position 42 by a leucine. Thepir42 mutation was described by Miron et al. (Proc Natl Acad Sci USA,1994. 91(14): p. 6438-42; EMBO J, 1992. 11(3): p. 1205-16), and wasreported to increase the copy number of an “ori gamma R6K-Km^(R)-pir42”plasmid by 2.5 fold as compared to the same plasmid harboring thewild-type pir gene. However the pir42 mutation was never used ordescribed in combination with the pir116 mutation and while othercopy-up mutations such as cop21 in the pir gene combined with the pir116do not exhibit an increase of the plasmid copy number, combination ofthe pir116 and pir42 mutations in a E. coli XAC-1 endA⁻ traD⁻ strainsurprisingly showed a significant increase of the plasmid copy number.Applicants have thus shown unexpected results of this combination interms of copy number of the plasmids produced in E. coli host strainscomprising the mutated pir116 and pir42 gene as compared with strainsharboring pir116 alone, or in a host cell comprising the pir116 mutationcombined with another mutation of the pir gene, such as the mutationcop21 (Inuzuka et al., FEBS Lett, 1988. 228(1): p. 7-11). For example,E. coli TEX1pir42 (=XAC-1 endA⁻ traD⁻ pir116 pir42) exhibited a 2-5 foldincrease in the number of plasmids, as compared to a pir116 strain, orstrains comprising combined pir116 and cop21 mutations (See Example 11).In other embodiments, the pir gene comprises at least one mutation,which, for example, may occur in the copy number control region, in theleucine zipper-like motif, in the DNA binding region, or in one or moreof these regions or another region of the protein pi coded by the pirgene.

The prokaryotic host cell according to the present invention alsocomprises one or more mutations in the same or a different domain of theprotein pi, coded by the pir gene copy, such as the DNA binding domain,and/or the copy number control region and/or the leucine-zipper motif.The prokaryotic recombinant host cell may comprise the heterologous pirgene is in a plasmid or in the genome of the host cell.

Such mutations may be screened by using the fluorescence-based screeningmethod according to one aspect of the present invention as describedthereafter. As shown in the Example 13, host cells comprising at leastone mutation in the pir gene, the mutation pir116 and a mutation in theDNA binding domain were screened using the fluorescence-based screeningmethod according to the present invention. Host cells comprisingmutations present in the DNA binding domain in addition to the pir116,i.e., as for example in the construct 100B, wherein the tyrosine (K) atposition 292 is replaced by a methionine (M), in the construct 114C,wherein a glutamic acid (E) at position 130 is replaced by a valine (V),or in the construct 201C, wherein an aspartic acid (D) at position 117is replaced by a glycine (G) (FIG. 26) are tested for their capacity toproduce high copy number of plasmid using the fluorescence-basedscreening method.

According to another embodiment of the present invention, the host cellused in the process claimed is a prokaryotic host cell in which the recAgene or a homologous gene has been inactivated. Preferably, the hostcell according to the present invention is E. coli strain XAC-1comprising mutations pir116, pir42, endA⁻, traD⁻¹ recap. Such a strainis designated TEX2pir42. recA may be inactivated by methods well knownto those in the art. recA encodes a major recombination protein andmutations in this gene reduce the frequency of recombination-mediatedalteration in plasmids and intramolecular recombination that could leadto the multimerization of plasmids. As described in Example 12, adeleted recA gene containing 3 translation stop codons (one in eachframe) at its 5′ end may be obtained by PCR. The resulting inactivatedgene was then introduced by gene replacement into TEX1 genome (Example12.1).

These cells are obtained by any technique known to those skilled in theart which allows the introduction of the said plasmid into a given cell.Such a technique may be, in particular, transformation, electroporation,conjugation, fusion of protoplasts or any other technique known to thoseskilled in the art.

Strain XAC-1pir116 was deposited under the terms of the Budapest Treatywith the Collection Nationale De Cultures de Micro-organismes (CNCM),Institut Pasteur, 28, rue Dr. Roux, 75724 Paris Cedex 15, France, onOct. 10^(th), 2003 under accession no. 1-3108.

Strain TEX2pir42 was deposited under the terms of the Budapest Treatywith the Collection Nationale De Cultures de Micro-organismes (CNCM),Institut Pasteur, 28, rue Dr. Roux, 75724 Paris Cedex 15, France, onOct. 10^(th), 2003, under accession no. I-3109.

The DNA molecules according to the invention may be used in anyapplication of vaccination or of gene and cell therapy for transferringa gene to a given cell, tissue or organism, or for the production ofrecombinant proteins in vitro.

In particular, they may be used for direct in vivo administration or forthe modification of cells in vitro or ex vivo, for the purpose ofimplanting them into a patient.

In this respect, another subject of the present invention relates to anypharmaceutical composition comprising at least one DNA molecule asdefined above. This molecule may or may not be associated therein with achemical and/or biochemical transfection vector. This may in particularinvolve cations (calcium phosphate, DEAE-dextran, etc.) or liposomes.The associated synthetic vectors may be cationic polymers or lipids.Examples of such vectors which may be mentioned are DOGS (Transfectam™)or DOTMA (Lipofectin™).

The pharmaceutical compositions according to the invention may beformulated for the purpose of topical, oral, parenteral, intranasal,intravenous, intramuscular, subcutaneous, intraocular, or transdermaladministrations. The claimed plasmid is preferably used in an injectableform or in application. It may be mixed with any vehicle which ispharmaceutically acceptable for an injectable formulation, in particularfor a direct injection to the site to be treated. This may involve, inparticular, sterile, isotonic solutions or dry compositions, inparticular freeze-dried compositions, which, by addition, depending onthe case, of sterilized water or of physiological saline, allowinjectable solutions to be made up. This may in particular involve Trisor PBS buffers diluted in glucose or in sodium chloride. A directinjection into the affected region of the patient is advantageous sinceit allows the therapeutic effect to be concentrated at the level of theaffected tissues. The doses used may be adapted as a function of variousparameters, and in particular as a function of the gene, the vector, themode of administration used, the pathology concerned or the desiredduration of the treatment.

The DNA molecules of the invention may contain one or more genes ofinterest, that is to say one or more nucleic acids (synthetic orsemi-synthetic DNA, gDNA, cDNA, etc.) whose transcription and, possibly,whose translation in the target cell generate products of therapeutic,vaccinal, agronomic or veterinary interest.

Among the genes of therapeutic interest which may be mentioned moreparticularly are genes coding for enzymes, blood derivatives, hormonesand lymphokines: interleukins, interferons, TNF, etc. (FR 92/03120),growth factors, neurotransmitters or their precursors or syntheticenzymes, and trophic factors (BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF,NT3, NT5, VEGF-B, VEGF-C etc.; apolipoproteins: ApoAI, ApoAIV, ApoE,etc. (FR 93/05125), dystrophin or a minidystrophin (FR 91/11947),tumour-suppressing genes: p53, Rb, Rap1A, DCC, k-rev, etc. (FR93/04745), genes coding for factors involved in coagulation: factorsVII, VIII, IX, etc., suicide genes: thymidine kinase, cytosinedeaminase, etc.; or alternatively all or part of a natural or artificialimmunoglobulin (Fab, ScFv, etc.), an RNA ligand (WO 91/19813), etc. Thetherapeutic gene may also be an antisense sequence or gene, whoseexpression in the target cell makes it possible to control theexpression of genes or the transcription of cellular mRNAs. Suchsequences may, for example, be transcribed, in the target cell, intoRNAs which are complementary to cellular mRNAs and thus block theirtranslation into protein, according to the technique described in patentEP 140,308. A insert of interest that may be carried by the pCOR of theinvention is a RNAi, whose is capable of interfering with thetranslation of a target gene (Wilson et al., Curr Opin Mol. Ther. 2003August; 5(4):389-96) and thereby regulating the expression of such gene.

The gene of interest may also be a vaccinating gene, that is to say agene coding for an antigenic peptide, capable of generating an immuneresponse in man or animals, for the purpose of producing vaccines. Theseantigenic peptides may in particular be specific antigenic peptides ofEpstein-Barr virus, HIV virus, hepatitis B virus (EP 185,573), orpseudorabies virus, or alternatively specific antigenic peptides oftumours (EP 259,212).

Generally, in the DNA molecules of the invention, the gene oftherapeutic, vaccinal, agronomic or veterinary interest also contains apromoter region for functional transcription in the target organism orcell, as well as a region located at the 3′ end which specifies atranscription termination signal and a polyadenylation site. As regardsthe promoter region, it may be a promoter region naturally responsiblefor expression of the gene under consideration when this region iscapable of functioning in the cell or the organism concerned. Thepromoter regions may also be regions of different origin (responsiblefor the expression of other proteins) or even of synthetic origin. Inparticular, they may be promoter sequences from eukaryotic or viralgenes. For example, they may be promoter sequences obtained from thegenome of the target cell. Among the eukaryotic promoters which may beused are any promoters or derived sequence which stimulates orsuppresses the transcription of a gene in a specific or non-specific,inducible or non-inducible, strong or weak manner. The eukaryoticpromoters may in particular be ubiquitous promoters (promoters of thegenes for HPRT, PGK, α-actin, tubulin, etc.), intermediate filamentpromoters (promoters of the genes for GFAP, desmin, vimentin,neurofilaments, keratin, etc.), therapeutic gene promoters (for examplethe promoters of the genes for MDR, CFTR, factor VIII, ApoAI, etc.)tissue-specific promoters (promoters of the genes for pyruvate kinase,villin, intestinal fatty acid-binding protein, α-actin of smooth muscle,etc.) or alternatively promoters which respond to a stimulus (steroidhormone receptor, retinoic acid receptor, etc.). Similarly, they may bepromoter sequences obtained from the genome of a virus, such as, forexample, the promoters of the adenovirus EIA and MLP genes, the CMVearly promoter or alternatively the LTR promoter of RSV, etc. Inaddition, these promoter regions may be modified by addition ofactivating or regulatory sequences or sequences which allowtissue-specific expression or expression which is predominantlytissue-specific.

Moreover, the gene of interest may also contain a signal sequence whichdirects the synthesized product into the secretory pathways of thetarget cell. This signal sequence may be the natural signal sequence ofthe synthesized product, but it may also be any other functional signalsequence or an artificial signal sequence. Preferred signal sequenceused according to the present invention is the secretion signal peptideof human interferon as described Taniguchi et al. (Gene, 1980, 233(4763):541-5)

Depending on the gene of interest, the DNA molecules of the inventionmay be used for the treatment or prevention of several pathologies,including genetic diseases (dystrophy, cystic fibrosis, etc.),neurodegenerative diseases (Alzheimer's disease, Parkinson's disease,ALS, etc.), cancers, pathologies associated with coagulation disordersor with dyslipoproteinaemias, pathologies associated with viralinfections (hepatitis, AIDS, etc.), or in the agronomic and veterinaryfields, etc.

According a preferred embodiment, the DNA molecules of the presentinvention are used for treating critical limb ischemia pathologies suchas for example peripheral arterial occlusive disease and intermittentclaudication.

Moreover, the present invention also relates to the use of conditionalreplication DNA molecules for the production of recombinant proteins.Bacteria can be used to produce proteins of various origins, eukaryoticor prokaryotic. Among the bacteria, E. coli constitutes the organism ofchoice for expressing heterologous genes on account of its ease ofmanipulation, the large number of expression systems available and thelarge amounts of proteins which can be obtained. It is understood thatthe system of the invention can be used in other organisms, the tropismbeing determined by the nature of the origin of replication, asindicated above. For this use, the nucleic acid sequence of interestcomprises a coding region under the control of expression signals thatare appropriate for the host chosen, in particular a prokaryotic host.These may be, for example, Plac, Ptrp, PT7, Ptrc, Ptac, PL, P_(BAD) orPR promoters, the Shine-Dalgarno sequence, etc. (this set constitutesthe expression cassette). The nucleic acid sequence of interest can beany sequence coding for a protein which is of value in the fields ofpharmacy, agri-foods, chemistry or agrochemistry. This may be astructural gene, a complementary DNA sequence, a synthetic orsemi-synthetic sequence, etc.

The expression cassette can be introduced onto the conditionalreplication vector which is the subject of the invention, thusconstituting a conditional replication vector which allows theexpression of proteins of interest in E. coli. This vector has severaladvantages: no use of antibiotic to select it in the bacterium (reducedcost, no need for a study regarding the presence of antibiotic or ofpotentially toxic derived products in the finished product), virtuallyno probability of dissemination of the plasmid in nature (conditionalorigin of replication), possible fermentation in entirely definedmedium. The examples given show the advantageous properties of theseconditional vectors for the production of recombinant proteins.

As described above, the DNA molecule according to the present inventioncomprises an origin of replication ORIγ derived from R6K wherein the pirgene is removed and is introduced into the genome of a specific hostcell that is used for the production of the DNA molecules at largescale. There is always a need to produce increasing quantities ofplasmid for clinical trials and/or for use in DNA-based gene therapy.Production host cells have been engineered to carry the pir genecontaining at least one mutation, such as the mutation pir116 and/orpir42. Use of such mutated host strain results in an increase of theplasmids copy number and thus significantly raises the yield ofproduction. Also, conformation of the plasmids so produced is verysatisfying.

According to a particular aspect, a novel fluorescence-based method ofscreening for copy-up mutant is provided. This fluorescence-basedscreening method is far superior to the classical method of screeningbased on the level of resistance to antibiotic in the bacteria, whichmay not be used when the basal copy number of plasmid is already veryhigh such as the one obtained using the mutant pir116, e.g., around 400copies of plasmid per cell. The fluorescence-based method of screeningaccording to the present invention preferably uses the cobA gene as redfluorescence reporter gene of copy-up number. The cobA gene which is agene from Pseudomonas denitrificans (Crouzet et al., J. Bacteriol. 1999,172: 5968-79) encodes uro III methyltransferase, an enzyme of thevitamin B12 pathway, which adds two methyl groups to urogen IIImolecule. Wildt et al. (Nature Biotechnology, vol. 17, 1999, pp1175) hasdescribed the use of cobA as a fluorescent transcriptional reporter genefor E. coli, yeast and mammalian cells. For example, such fluorescentreporter gene was used for the selection of recombinant plasmidscontaining E. coli strains which accumulate fluorescent porhyrinoidcompounds due to overexpression of the cobA gene encoding the uroIIImethyltransferase. When illuminated with UV light, the cells fluorescedwith a bright red color (Biotechniques, 1995, vol 19, no. 7, p. 760).

The Applicant has surprisingly found a close correlation between thecopy number of plasmid carrying the cobA gene and the level offluorescence from pink to red. The fluorescence-based method ofscreening of copy-up mutants according to the present invention is thususeful for screening various mutants which can then be evaluated in thegenome of the production host cell, such as E. coli, or mutants of anygenes such as in the pir gene, which are inserted in the genome of theproduction host cell or carried in a plasmid.

In addition to the correlation with the copy number of plasmids, thefluorescence-based method of screening of the present invention iseasily and rapidly conducted as it is only requires plating andculturing the transformed host cells overnight and exposing to UVlights, to reveal intensity of the fluorescence produced, therebydeducing directly the number of copy of plasmids in the host cell.

Thus, the present invention provides for a method for detecting aplasmid copy-up mutation comprising:

-   -   (a) introducing at least one mutation into a target sequence;    -   (b) transforming the mutated target sequence into a host cell        comprising a plasmid, wherein the plasmid comprises a nucleotide        sequence encoding uroIII methyltransferase and the copy number        of the plasmid is effected by the target sequence;    -   (c) growing the host cell under conditions wherein the        nucleotide sequence is expressed to produce a culture of host        cells;    -   (d) exposing the culture of host cells to UV light; and    -   (e) detecting fluorescence produced by the culture of host        cells.

According to the present invention, the method further comprisescomparing the fluorescence detected in (e) with fluorescence produced bya culture of host cells comprising an non-mutated target sequence.

Preferably, the uroIII methyltransferase gene is coded by the cobA genefrom Pseudomonas denitrificans.

The mutation may be present in a plasmid comprising a heterologous pirgene comprising at least one mutation. The plasmid may comprise at leastone mutation in the pir other regions such as in the copy control regionand/or in the DNA binding domain, and/or in the leucine-zipper motifand/or in another region of the pir gene. Also, the plasmid may compriseat least one mutation in the heterologous pir gene copy number controlregion and the leucine zipper-like motif. The plasmid may furthercomprise a mutation in the pir gene DNA binding region. Furthermore, theplasmid may comprise one or more mutations in the same or a differentregion of the pir gene coding for the copy control region and/or the DNAbinding region, and/or the leucine zipper-like motif, or other region ofthe protein H.

Within limitation, the prokaryotic recombinant host cell according tothe present invention comprises the pir116 mutation and a secondmutation in the DNA binding region such aspir292, pir130, or pir117(FIG. 26).

Such mutated production host strain may be advantageously produced usingan universal plasmid tool such as the minicircle. The minicircletechnology is described inter alia in U.S. Pat. Nos. 6,143,530 and6,492,164 of the Applicant or in PCT application WO 96/26270.

Minicircles are recombinant DNA molecules that do not contain any originof replication, and thus represent excellent suicide vector for genereplacement of the genome of any microorganisms. In particular, the geneor genes of interest are flanked by the two sequences permittingsite-specific recombination, positioned in the direct orientation in theminicircle. The position in the direct orientation indicates that thetwo sequences follow the same 5′-3′ polarity in the recombinant DNAminicircle. The minicircle genetic constructions are generally circulardouble-stranded DNA molecules devoid of origin of replication, but mayalso be in linear form and contain the gene or genes of interest flankedby the two sequences permitting site-specific recombination, positionedin the direct orientation. According to this particular embodiment ofthe invention, the minicircle may be used to transform any competentmicroorganisms for the purpose of the gene replacement within the genomethereof (FIG. 31).

The minicircle for gene replacement is generated from a parent plasmidcomprising at least:

a) an origin of replication and, a selection marker gene,

b) two sequences permitting site-specific recombination, positioned inthe direct orientation, and,

c) placed between said sequences b), one or more genes of interest.

The specific recombination system present in the genetic constructionscan be of different origins. In particular, the specific sequences andthe recombinases used can belong to different structural classes, and inparticular to the integrase family of bacteriophage λ or to theresolvase family of the transposon Tn3. Among recombinases belonging tothe integrase family of bacteriophage λ, there may be mentioned, inparticular, the integrase of the phages lambda (Landy et al., Science197: 1147, 1977), P22 and (D80 (Leong et al., J. Biol. Chem. 260: 4468,1985), HP1 of Haemophilus influenza (Hauser et al., J. Biol. Chem. 2676859, 1992), the Cre integrase of phage P1, the integrase of the plasmidpSAM2 (EP 350,341) or alternatively the FLP recombinase of the 2μplasmid. The minicircles are thus prepared by recombination by means ofa site-specific system of the integrase family of bacteriophage λ, theDNA molecules according to the invention generally comprise, inaddition, a sequence resulting from the recombination between two attattachment sequences of the corresponding bacteriophage or plasmid.

Among recombinases belonging to the family of the transposon Tn3, theremay be mentioned, in particular, the resolvase of the transposon Tn3 orof the transposons Tn21 and Tn522 (Stark et al., Trends Genet, 8,432-439, 1992); the Gin invertase of bacteriophage mu, or,alternatively, the resolvase of plasmids, such as that of the parfragment of RP4 (Albert et al., Mol. Microbiol. 12: 131, 1994). When theminicircles are prepared by recombination by means of a site-specificsystem of the family of the transposon Tn3, they generally comprise, inaddition to the gene of interest that is aimed to be inserted in amicroorganism genome, a sequence resulting from the recombinationbetween two recognition sequences of the resolvase of the transposon inquestion. Sequences permitting site-specific recombination may also bederived from the loxP region of phage P1, which is composed essentiallyof two repeat sequences capable of recombining specifically with oneanother in the presence of a protein, designated Cre (Sternberg et al.,J. Mol. Biol. 150: 467, 1971). The plasmid used to produce theminicircle thus comprises (a) a bacterial origin of replication and, aselection marker gene; (b) the repeat sequences of bacteriophage P1(loxP region); and (c), placed between said sequences (b), one or moregenes of interest that one's wish to insert in a microorganism genome.

Minicircles may comprise sequences permitting site-specificrecombination are derived from a bacteriophage, such as attachmentsequences (attP and attB sequences) of a bacteriophage or sequencesderived from such attachment sequences. These sequences are capable ofrecombining specifically with one another in the presence of arecombinase referred to as an integrase with or without an excisionase.The term “sequences derived from such attachment sequences” includes thesequences obtained by modification(s) of the attachment sequences of thebacteriophages that retain the capacity to recombine specifically in thepresence of the appropriate recombinase. Thus, such sequences can bereduced fragments of these sequences or, alternatively, fragmentsextended by the addition of other sequences (restriction sites, and thelike). They can also be variants obtained by mutation(s), in particularby point mutation(s). The terms attP and attB sequences of abacteriophage or of a plasmid denote, according to the invention, thesequences of the recombination system specific to said bacteriophage orplasmid, that is to say the attP sequence present in said phage orplasmid and the corresponding chromosomal attB sequence. Attachmentsequences are well known in the art, and include inter alia theattachment sequences of the phages λ, P22, Φ80, P1, and HP1 ofHaemophilus influenzae or, alternatively, of plasmid pSAM2 or the2plasmid.

The minicircles are easily produced from the parent plasmid describedabove. The method for the production of the minicircle consists inbringing into contact culture of cells that are transformed with theparent plasmid with the integrase with or without the excisionase, so asto induce the site-specific recombination. The culture and the integrasewith or without the excisionase are brought into contact either bytransfection or infection with a plasmid or a phage containing the genefor said integrase and when applicable the gene for the excisionase.Alternatively, for example, the expression of genes coding for saidintegrase and when applicable the excisionase, present in the host cell,are induced. As mentioned below, these genes may be present in the hostcell in integrated form in the genome, on a replicative plasmid, or,alternatively, on the plasmid of the invention, in the non-therapeuticportion.

To permit the production of the minicircles according to the inventionby site-specific recombination in vivo, the integrase with/without theexcisionase used are introduced into, or induced in, cells or theculture medium at a particular instant. For this purpose, differentmethods may be used. According to a first method, a host cell is usedcontaining, for example, the recombinase gene, i.e., the integrase genewith or without the excisionase gene, in a form permitting its regulatedexpression. The integrase gene with or without the excisionase gene may,for example, be introduced under the control of a promoter, or of asystem of inducible promoters, or, alternatively, in atemperature-sensitive system.

In particular, the integrase gene may be present in atemperature-sensitive phage, latent during the growth phase, and inducedat a suitable temperature (for example, lysogenic phage λ Xis⁻ c1857).

Alternatively, the gene may be under the control of a regulatedpromoter, for example, the placUV5 promoter, the host cell is designatedE. coli G6191.

Preferably, the integrase with or without the excisionase gene may beunder the control of a regulated promoter, for example the P_(BAD)promoter of the araBAD (arabinose) operon, which is regulated byarabinose (Guzman et al., J. Bacteriol, 1995, 4121-4130; U.S. Pat. No.5,028,530). Particularly, use of P_(BAD) promoter allows sufficientexpression of excisionase and integrase in presence of arabinose, as theinducing agent, and thus more than 90% of recombination of the plasmidswhich are present in high copies number in the bacteria, whereas inabsence of arabinose, the promoter is tightly inhibited. The cassettefor expression of the integrase with/without excisionase may be carriedby a plasmid, a phage, or even by the plasmid of the invention in thenon-therapeutic region. It may be integrated in the genome of the hostcell or maintained in replicative form. Such host cells are inparticular E. coli G6264 and E. coli G6289. According to another method,the cassette for expression of the gene(s) is carried by a plasmid or aphage used to transfect or infect the cell culture after the growthphase. In this case, it is not necessary for the gene to be in a formpermitting its regulated expression. In particular, any constitutivepromoter may be used. The DNA may also be brought into contact with theintegrase and when applicable the excisionase in vitro, on a plasmidpreparation, by direct incubation with the protein.

The minicircle so produced thus comprises an expression cassettecontaining one or more genes of interest to be inserted in the targetedmicroorganism, lacks an origin of replication and comprises a sequenceattR resulting from site-specific recombination between an attB and anattP sequence, or a sequence attL resulting from site-specificrecombination between an attB and an attP sequence. The minicircle maythus be used as universal suicide vector for gene replacement in anymicroorganisms. In effect, the minicircle carrying a gene forreplacement flanked by homologous sequences and a antibiotic resistancegene will easily integrate in a targeted site of the genome of anymicroorganism by homologous recombination as represented in FIG. 31. Asecond event of excision which may be triggered by a second selectionpressure may then efficiently select the microorganisms only carryingthe new inserted gene within their genome.

The present invention thus also relates to a method of gene engineeringof a microorganism. This novel method may used to engineer anymicroorganism regardless of their origin. In effect, the minicircle doesnot contain any origin of replication, and thus can be used universallyfor gene replacement in any types of microorganisms. This methodrepresents an advantageous alternative to the use of the bacteriophageM13 for gene replacement by double homologous recombination in amicro-organism.

According to a particular embodiment of the present invention, theminicircle comprises a first selectable marker such as an antibioticresistance gene, allowing selecting for the first recombination event.Preferred second selectable marker is the gene III or the functionaldeleted gene III′. The gene III or its functional variant is capable ofconferring sensibility to deoxycholate as described in Boecke et al.(Mol. Gen. Genet., 186, 185-92, 1982) and thus allows forcounter-selecting the second event of recombination (FIG. 31). Themethod thus consists in introducing the minicircle into themicroorganism by any transformation method well known in the art, andpreferably by electroporation, selecting the event of integration of theminicircle in a culture supplemented with an antibiotic or under anotherpression of selection, and selecting a second event of excision bytreating with deoxycholate or another appropriate pression of selection.

The present invention will be described more fully with the aid of theexamples which follow, which should be considered as non-limitingillustrations.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Functional organization of the region of R6K involved inreplication.

FIG. 2: Organization of the functional domains of the Π protein of theplasmid R6k.

FIG. 3: Representation of the protocol for introducing the pfr gene intothe genome of E. coli XAC 1.

FIG. 4: Construction scheme for vectors pXL2666, 2730 and 2754.

FIG. 5: Construction of pXL2774.

FIG. 6: Growth and production kinetics in a 2 L fermenter.

FIG. 7: Growth and production kinetics in an 800 L fermenter.

FIG. 8: Construction of pXL3056.

FIG. 9: Visualization of the aFGF protein produced by E. coliXAC-1pir116 (pXL3056+PT7pol23) after induction. The denatured total cellextracts are deposited on 12.5%-SDS polyacrylamide gel. M: molecularmass marker (Biorad, Low range). Each band is identified by an arrow anda figure which indicates its mass in kDaltons. 1: XAC-1pir116(pXL3056+pUC4K) not induced; 2: XAC-1pir116 (pXL3056+pUC4K) induced at42° C.; 3: XAC-1pir116 (pXL3056+PT7pol23) clone 1, not induced; 4:XAC-1pir116 (pXL3056+PT7pol23) clone 1, induced at 42° C.; 5:XAC-1pir116 (pXL3056+PT7pol23) clone 2, not induced; 6: XAC-1pir116(pXL3056+PT7pol23) clone 2, induced at 42° C.; t1: 1 μg of purifiedaFGF; t4: 4 μg of purified aFGF.

FIG. 10: Schematic representations for vectors pXL3029, pXL3030, andpXL3179 or NV1FGF.

FIG. 11: Schematic representation of the functional domains of R6K πinitiator proteins.

FIG. 12: Nucleotide (SEQ ID NO: 21) and amino acid (SEQ ID NO: 22)sequences of the pir gene comprising the pir116 and pir42 mutations.

FIG. 13: Construction of pir116pir42 suicide vector for homologousrecombination.

FIG. 14: Schematic representation of the PCR products obtained whenamplifying the region uidA::pir116+/−pir42.

FIG. 15: Agarose gel electrophoresis showing the topology of pCORplasmid pXL3179 produced in TEX1 or TEX1pir42.

FIG. 16: Schematic representation of the pXL3749 suicide plasmidcarrying pir116cop21 gene.

FIG. 17: Agarose gel electrophoresis showing the plasmid copy number ofpXL2979 when produced in E. coli host cell TEX1cop21 (lines 1-4), in E.coli host cell XAC1pir (lines 5-8), in E. coli TEX1 (lines 9-12).

FIG. 18: Representation of the cloning strategy for the construction ofthe recA-suicide vector.

FIG. 19: Schematic representation of the PCR products obtained whenamplifying regions of E. coli TEX2 strain. FIG. 20: Agarose gelelectrophoresis showing the topology of pCOR pXL3179 produced in E. coliTEX2pir42 (line B), in E. coli TEX1pir42 (line C), in E. coli TEX1 (lineD).

FIG. 21: Analysis of plasmid pXL3179 produced by fermentation in E. coliTEX2pir42.

FIG. 22: Fluorescence-based assay showing that fluorescence increaseswith plasmid copy number.

FIG. 23: Diagram of plasmids screened in the fluorescence-based assay.

FIG. 24: Diagram of plasmid pXL3830.

FIG. 25: Agar plate demonstrating fluorescence-based screening forcopy-up mutants generated by random mutagenesis.

FIG. 26: Evaluation of copy-up mutants identified by thefluorescence-based screening method.

FIG. 27: Diagram of the strategy for evaluating pir116 mutants insertedinto the bacterial genome.

FIG. 28: Evaluation of pXL3179 copy number in different pir116* mutantE. coli strains.

FIG. 29: Construction of a plasmid used to generate minicircle vectorsfor homologous recombination in E. coli.

FIG. 30: Construction of a minicircle vector used to generate pir116*mutant E. coli strains.

FIG. 31: Diagram of gene replacement by homologous recombination using aminicircle vector.

FIG. 32: Demonstration of double recombinant clones grown on mediumcontaining sodium deoxycholate.

FIGS. 33A and B: The results of control PCR on double recombinants.

I Materials and Methods A) Materials

1) Culture Media

Complete LB, 2×TY and SOC media and minimal M9 medium (Maniatis et al.,1989) were used. Agar media were obtained by addition of 15 g of Difcoagar. Furthermore, if necessary, these media were supplemented withantibiotics (ampicillin or kanamycin) at respective concentrations of100 mg/l and 50 mg/l. The chromogenic substrates X-Gal and X-Gluc wereused at a concentration of 40 mg/l.

2) E. coli Strains, Plasmids and Bacteriophages

The E. coli strains, plasmids and bacteriophages used are respectivelyidentified in the examples below.

B) Methods 1) Manipulation of the DNA

The isolation of bacterial DNA (plasmid and genomic) and phage DNA(replicative form of M13), digestion with restriction endonucleases,ligation of the DNA fragments, agarose gel electrophoresis (in TBEbuffer) and other standard techniques were carried out according to themanufacturers' recommendations, for the use of enzymes, or in accordancewith the procedures described in “Molecular Cloning: a LaboratoryManual” (Maniatis et al., 1989).

The DNA size markers used during the electrophoreses are as follows: 1kb ladder (BRL) for the linear fragments and the supercoiled DNA marker(Stratagene) for the undigested plasmids.

Sequencing was carried out according to the Sanger technique (Sanger etal., 1977) adapted to the automated method using fluorescentdideoxynucleotides and Taq DNA polymerase (PRISM Ready ReactionDyeDideoxy Terminator Cycle Sequencing Kit, Applied Biosystems).

The oligodeoxynucleotides used (designated by “seq+no.”, see below) weresynthesized on the “Applied Biosystems 394 DNA/RNA Synthesizer” by thephosphoramidite method, using α-cyanoethyl protecting groups (Sinha etal., 1984). After synthesis, the protecting groups are removed bytreatment with ammonia. Two precipitations with butanol allow theoligonucleotide to be purified and concentrated (Sawadogo et al., 1991).

Sequences of the Oligonucleotides used for the PCR Amplification:

SEQ ID No. 3 5′-GACCAGTATTATTATCTTAATGAG-3′ SEQ ID No. 45′-GTATTTAATGAAACCGTACCTCCC-3′ SEQ ID No. 55′-CTCTTTTAATTGTCGATAAGCAAG-3′ SEQ ID No. 65′-GCGACGTCACCGAGGCTGTAGCCG-3′

The PCR reactions (Safki et al., 1985) were performed under thefollowing conditions, in a total volume of 100 μl. The reaction mixturecomprises 150 ng of genomic DNA from the strain to be studied, 1 μg ofeach of the two oligonucleotide primers (24-mer), 10 μl of 10×PCRbuffer, the composition of which is as follows “500 mM KCl, 0.1%gelatin, 20 mM MgCl₂, 100 mM Tris-HCl pH 7.5”, and 2.5 units of Taq DNApolymerase (Amplitaq Perkin-Elmer). The PCR conditions, on thePerkin-Elmer Cetus DNA Thermal Cycler machine are as follows: 2 min at91° C., 30 successive cycles of denaturation (1 min at 91° C.),hybridization (2 min at 42° C.) and elongation (3 min at 72° C.), andfinally 5 min at 72° C. The products thus obtained, which are or are notdigested with a restriction enzyme, are analysed by agarose gelelectrophoresis.

Analysis of the various plasmid species by DNA topoisomerases wasperformed according to the following procedure: the enzymes, purified inthe laboratory, are incubated for 1 hour at 37° C. The reaction mixtures(total volume: 40 μl) have the following composition: 150 ng of plasmid,300 ng of DNA topoisomerase 1 or 150 ng of E. coli DNA gyrase, or 160 ngof S. aureus DNA topoisomerase IV and 20 μl of buffer specific for eachenzyme. The composition of these buffers is indicated below:

for DNA topoisomerase I:50 mM Tris-HCl pH 7.7, 40 mM KCl, 1 mM DTT, 100 μg/ml BSA, 3 mM MgCl₂, 1mM EDTA; for DNA topoisomerase IV:60 mM Tris-HCl pH 7.7, 6 mM MgCl₂, 10 mM DTT, 100 μg/ml BSA, 1.5 mM ATP,350 mM potassium glutamate;for DNA gyrase:50 mM Tris-HCl pH 7.7, 5 mM MgCl₂, 1.5 mM ATP, 5 mM DTT, 100 μg/ml BSA,20 mM KCl.

2) Transformation of E. coli

This was performed routinely according to the TSB (Transformation andStorage Buffer) method described by Chung and Miller (1988). For astrain such as TG1 (Gibson et al., 1984), the transformation efficiencyobtained is about 10⁵-10⁶ transformants per μg of pUC4K (Vieira andMessing; 1982). When a higher transformation efficiency was necessary,the bacteria were transformed by electroporation according to theprocedure recommended by the electroporator manufacturer (Biorad). Thismethod makes it possible to achieve efficiencies of from 10⁸ to 10¹⁰transformants per μg of pUC4K.

3) Cellular Transfection Mediated by a Cationic Lipofectant

The cells used are NIH 3T3 mouse fibroblasts seeded the day before into24-well plates, at a density of 50,000 cells per well. The culturemedium used is DMEM medium, containing 4.5 g/l of glucose andsupplemented with 10% fetal calf serum and 1% of solutions of 200 mMglutamine and antibiotics (5.10³μ/ml streptomycin, 5.10³ μg/mlpenicillin) (Gibco). The plasmid DNA (1 μg in 25 μl of 9% NaCl) ismixed, on a volume-for-volume basis, with a suspension of lipofectant.Four “lipofectant charges/DNA charges” ratios are tested: 0, 3, 6 and 9.These ratios are calculated by considering that 1 μg of plasmid DNAcarries 3.1 nmol of negative charges and that the lipofectant contains 3positive charges per molecule. After a contact time of 10 minutes toallow formation of the DNA/lipid complex, 50 μl of DNA-lipofectantmixture are introduced onto the cells in serum-free culture medium (500μl). The cells were prerinsed twice with this same medium. Inhibition oftransfection by the serum is thus avoided. After incubation (2 hours at37° C. in the CO₂ incubator), 10% fetal calf serum is added to themedium. The cells are then reincubated for 24 hours.

4) Measurement of the Luciferase Activity of Eukaryotic Cells

This is carried out 24 hours after the transfection. Luciferasecatalyses the oxidation of luciferin in the presence of ATP, Mg²⁺ andO₂, with concomitant production of a photon. The total amount of lightemitted, measured by a luminometer, is proportional to the luciferaseactivity of the sample. The reagents used are supplied by Promega(luciferase assay system) and used according to the recommendedprocedure. After lysis of the cells, the insoluble fraction from eachextract is eliminated by centrifugation. The assay is carried out on 5μl of supernatant, which may or may not be diluted in the cell lysisbuffer.

5) Measurement of the Protein Concentration in the Cell Extracts

This is carried out according to the BCA method (Pierce) usingbicinchoninic acid (Wiechelman et al., 1988). The standard BSA range isprepared in the lysis buffer (cf. III-B-4). The samples to be assayedand those of the range are pretreated, on a volume-for-volume basis,with 0.1 M iodoacetamide/0.1 M Tris buffer, pH 8.2, for 1 hour at 37° C.This treatment makes it possible to prevent interference, during theassay, of the reducing agent (DTT) present in the lysis buffer. Theassay result is read at 562 nm.

EXAMPLE 1 Construction of XAC-1 pir and pir116 Host Strains byHomologous Recombination

The strain used was the E. coli strain XAC-1 (Normanly et al., 1980).The argE gene of this strain advantageously includes a mutation ofglutamine-53 (CAG) into the amber codon (TAG) (Meinnel et al., 1992).The argE gene belongs to the argECBH divergent operon and codes for anarginine biosynthesis enzyme, N-acetylornithinase. XAC-1 cannottherefore synthesize arginine and, consequently, grow in minimal medium.This auxotrophy will be relieved if the strain harbors a plasmid whichallows the expression of a suppressor tRNA. It will thus be possible, byculturing in minimal medium, to select bacteria that carry such aplasmid. In order to allow the replication therein of plasmids derivedfrom R6K, it was necessary to introduce, by homologous recombination,the pir gene into the genome of XAC-1. The pir gene (wild-type ormutated) is introduced at the uidA locus by exchange between thewild-type uidA gene and a copy interrupted by the pir (or pir116) gene.The uidA gene codes for β-glucuronidase, the enzyme for hydrolysis ofβ-glucuronides. This gene may be inactivated without any problem sinceit is not essential for growth in standard synthetic media, in whichβ-glucuronides are not used. Furthermore, the β-glucuronidase activitycan be monitored by means of a chromogenic substrate, X-Gluc, whosehydrolysis releases a blue pigment.

1) Construction of a Suicide Vector Carrying the Cassette“Km^(R)-uidA::pir (or pir116)

We used a strategy involving a single bacterial host and minimizing themodifications to the genome of the strain of interest. The phage M13mp10 (Messing et Vieira; 1982) was used as a suicide vector (Blum etal., 1989). An amber mutation in the gene II, which is essential forreplication, reduces the host spectrum of this M13 to the strains, suchas TG1 (supE), which produce an amber suppressor tRNA; it will thereforenot be able to replicate in E. coli sup+ strains, such as XAC-1.

The 3.8 kb BamHI cassettes, containing the kanamycin-resistance gene ofTn5 and _uidA::pir or pir116, were respectively purified from M13wm34and 33 (Metcalf et al., 1994). They were cloned into M13 mp10 linearizedwith BamHI. The recombinant clones were selected by plating on LB agarmedium supplemented with kanamycin, after electroporating the ligationmixtures into TG1. The conformity of the clones obtained was shown byanalysing the restriction profile and by sequencing the regioncorresponding to the pir116 mutation.

2) Introduction of the pir or pir116 genes into the genome of E. coliXAC-1 by homologous recombination

The strategy adopted and the various events involved are presented inFIG. 3.

a) First Recombination Event

The XAC-1 strain was transformed by electroporation with 10, 100 or 2000ng of each RF (mp10-_uidA::pir or pir116). One-third of each expressionmixture was plated out on LB plates containing kanamycin and incubatedovernight at 37° C. The mp10-_uidA::pir or pir116 phages cannotreplicate in the strain XAC-1 (sup+). The kanamycin resistance(“Km^(R)”) marker can therefore only be maintained by integration intothe genome of the bacterium via a homologous recombination with thewild-type copy of the gene uidA. The results of the electroporations ofXAC-1 are presented in Table 1. The transformation efficiency obtainedwas 4.10⁹ transformants per μg of pUC4K.

TABLE 1 Number of colonies obtained with the amounts of DNA transformedCONSTRUCT 10 ng 100 ng 2000 ng M13mp10-_uidA::pir 1 41 146M13mp10-_uidA::pir116 0 16 124

Under the test conditions, the number of integrants increased in anon-linear manner with the amount of DNA. Given the transformationefficiency and the size of the RFs (11.7 kbp), it was possible to havean approximate idea of the level of recombination. By considering thepoint at 100 ng, a recombination frequency of about 1106 was obtained.

b) Second Recombination Event

The second recombination event will then be selected by the resistanceof the strains to deoxycholate (“Doc R”).

To do this, five integrants of each construct were cultured in 2XTYmedium supplemented with 0.2% sodium deoxycholate. Two distinctpopulations appeared. Certain clones gave quite visible cloudiness afterabout 8 hours at 37° C. (two clones for the pir construction and threefor the pir116 construction). The other clones gave a dense culture onlyafter one night at 37° C. They were virtually all sensitive to kanamycin(“Km^(s)”), as expected. For each of the electroporants studied, 50Km^(S) descendants were streaked onto LB medium supplemented withX-Gluc. After 48 hours at 37° C., the UidA⁺ clones were pale bluewhereas those which had undergone an allele replacement (case No. 1,FIG. 3) remained white on this medium (UidA⁻). Table 2 summarizes thephenotypic analysis of the double recombinants obtained. From 18 to 30%of the double recombinants underwent an allele replacement.

TABLE 2 Number of Km^(S) Percentage of UidA⁻ Strain among the Doc^(R)among the Km^(S) XAC-1 pir-2 50/50 18 XAC-1 pir-3 50/50 24 XAC-1 pir-450/50 34 XAC-1 pir116-1 50/50 32 XAC pir116-4 35/50 30

Checking the Pir+ character nature of the strains obtained byrecombination

To ensure the Pir+ character of the strains obtained by doublerecombination, we transformed three clones of each construct with pBW30(Metcalf et al., 1994). The fact that transformants were obtained forall the test strains made it possible to show the functionality of thepir and pir116 genes, which were integrated into the genome of XAC-1.Under the same conditions, no transformant was obtained with theparental strain XAC-1. We continued to study two XAC-1pir clones (B andC) and two XAC-1pir116 clones (E and D).

4) Checking by PCR Amplification of the Strains Obtained byRecombination

To confirm the allele replacement, we checked the genomic regions oneither side of the uidA locus by PCR amplification. Each pair ofoligonucleotides consisted of an oligonucleotide corresponding to aninternal region of pir and a second oligonucleotide corresponding to aregion, close to chromosomal uidA, but not within the fragment whichserved for the recombination. The sequence of the latter oligonucleotidewas determined by means of the ECOUIDAA sequence from Genbank (accessnumber: M14641). We were thus able to verify the exact location of thepir gene in the bacterial genome. The nature of the amplified fragments,whose size is in accordance with that which might be expected, wasconfirmed by digestion with MluI.

EXAMPLE 2 Construction of Plasmid Vectors Derived from R6K Carrying theSelection Marker sup Phe

Vectors were constructed containing ori γ from R6K and thekanamycin-resistance gene (pXL2666). The observation of pXL2666multimers in the strain BW19610 (pir116) 5 (Metcalf et al., 1993) led usto study the effect of the cer fragment from ColE1 on this phenomenon.We then introduced the expression cassette of the phenylalaninesuppressor tRNA (sup Phe) onto the vector ori γ-Km^(R)-cer (pXL2730).This vector, pXL2760, serves as a basis for the construction of vectorswhich can be used in gene therapy.

1) Construction and Analysis of Vectors Containing ori γ from R6K andthe Kanamycin Resistance Gene

a) Constructs

In the first plasmid constructed, pXL2666, the kanamycin resistance geneoriginated from pUC4K (Vieira and Messing; 1982) and the origin ofreplication, contained in a 417 bp EcoRI-BamHI fragment, originated fromthe suicide vector pUT-T7pol (Herrero et al., 1990) (FIG. 4). Thetransfer of pXL2666 into the strains BW 19094 and 19610 (Metcalf et al.,1994) made it possible to show that the amount of plasmid is indeedincreased in a pir116 strain, when compared with the same plasmid in apir strain. However, electrophoretic analysis of the undigested plasmidsshowed that this increase goes hand in hand with the appearance of a fewmultimeric forms. This phenomenon is quite probably associated withintermolecular recombination between the multiple copies of the plasmid.Thus, we constructed pXL2730 by cloning the cer fragment of the naturalE. coli plasmid, ColE1, which had been shown to permit, in cis, theresolution of plasmid dimers (Summers and Sherrat, 1984), into pXL2666.The fragment used corresponds to a 382 bp HpaII fragment from ColE1(Leung et al., 1985). It contains a specific intermolecularrecombination site; in order to function, it involves only host proteinsincluding the recombinases XerC and XerD and the accessory factors ArgRand PepA (Stirling et al., 1988, 1989; Colloms et al., 1990). To ensurethat the effects observed are indeed due to the cer fragment, we alsoconstructed the control plasmid pXL2754, in which the cer fragment has a165 bp deletion. This deletion was shown to abolish the action of cer onthe resolution of the multimers (Leung et al., 1985). The variouscloning steps leading to the construction of these plasmids arepresented in FIG. 4.

b) Quantitative and Qualitative Analysis of the Plasmid Species

(i) analysis by Agarose Gel Electrophoresis

Electrophoretic analysis of the different plasmids constructed allowedthe demonstration of various plasmid species, which are variableaccording to the strains used. The size of the undigested plasmids wasevaluated relative to a supercoiled DNA marker. In the pir strain(BW19094), the plasmids pXL2666, 2754 and 2730 were almost entirely inmonomeric form. The bands above each main band correspond to variousslightly less supercoiled topoisomers, as confirmed by the profileobserved after the action of DNA gyrase on pXL2730.

In the case of the pir116 strain (BW19610), the profiles were different:with the plasmids pXL2666 and 2754 different species were observedranging from the monomer to multimers (2, 3 or 4 units), the major formbeing the dimer. After digestion with EcoRI, only the linear plasmid DNAwas found; these plasmid species correspond either to plasmid multimersor to various topoisomers. However, since the size of the formsdetermined according to the supercoiled DNA marker was a whole productof that of the monomer plasmid, it is highly probable that they aremultimers. The formation of multimers was most probably attributable tothe pir116 mutation, although the two strains BW19094 and BW19610 arenot strictly isogenic (BW19610 is recA). The profile obtained withpXL2730 was different: although multimeric forms were still visible, themajor form is the monomeric form. The cer fragment can thus facilitateresolution of the plasmid multimers which we have constructed,independently of recA, in BW19610.

(ii) analysis after Treatment with DNA Topoisomerases

To disprove the theory that the forms observed in the strains carryingthe pir116 allele are specific topoisomers, each plasmid preparation wassubjected to the action of DNA topoisomerases. The activities of thevarious enzymes under the experimental conditions were as follows:relaxing of DNA for E. coli DNA topoisomerase I, negative supercoilingof relaxed DNA for E. coli DNA gyrase, and disentanglement of interlacedDNAs and relaxation of supercoiled DNA by S. aureus DNA topoisomeraseIV. The action of DNA topoisomerase IV made it possible to show that thehigh-molecular-weight plasmid forms did not result from the entanglementof several plasmid molecules; in this case, they would then have beenconverted into the monomeric species. The functionality of the enzymewas, of course, checked on a preparation of kinetoplast DNA, composed ofentangled DNA molecules (not shown). The relaxation activity was alsovisible since species are obtained which migrate less than in theuntreated controls. The action of DNA gyrase made it possible to convertthe slightly relaxed topoisomers into the more supercoiled speciesextracted from the bacterium (monomer or dimer mainly). Furthermore, itmade it possible to verify that the DNAs prepared were mainly insupercoiled form. The samples thus treated allowed the above results tobe confirmed as regards the major species for each construct. DNAtopoisomerase I did indeed relax DNA, but only partially. This could bedue to the fact that the plasmids studied contain only a fewsingle-stranded regions, to which this enzyme preferably binds (Roca,1995).

2) Introduction of the Selection Marker sup Phe into pXL2730

We used the expression cassette of the synthetic suppressor tRNA gene(Phe) (Kleina et al., 1990). This introduced a phenylalanine into thegrowing polypeptide chain in response to a TAG codon. Furthermore, itallowed the production in XAC-1 of an ArgE protein that was sufficientlyactive to allow good growth in arginine-deficient medium. sup Phe wasexpressed constitutively on the plasmid pCT-2-F (Normanly et al., 1986)from a synthetic promoter derived from the promoter sequence, Plpp, ofthe E. coli lpp gene. Downstream of this gene, transcription was stoppedby the synthetic terminator, T_(rrnC), of the E. coli operon rrnC(Normanly et al., 1986). The various cloning steps are indicated in FIG.5.

The various subclonings were performed in XAC-1. The functionality ofthe suppressor tRNA expression cassette was thus checked by means of theα-galactosidase activity of this strain, which only exists if there issuppression of the amber codon of the gene lacZ_(u118am). The final stepconsists of the introduction of the sup Phe expression cassette intopXL2730. The results obtained with the cer fragment (B-1-b) led us toselect this plasmid rather than pXL2666. We retained the kanamycinresistance gene for ease of subsequent cloning, in particular in orderto have available additional screening during the final cloning (loss ofKm^(R)).

EXAMPLE 3 Validation of the Plasmid Vector for Applications in GeneTherapy by Transfection of Mouse Fibroblasts

1) Construction of the Reporter Vector pXL2774

To test the validity for gene therapy of the system for producingplasmid DNA, we introduced a reporter gene, which can be used ineukaryotic cells, into pXL2760. We used the gene luc, which codes forPhotinus pyralis luciferase, since the bioluminescence measurement testis very sensitive and is linear over a large range, and the backgroundnoise due to the endogenous activity of eukaryotic cells is very low.The luc gene was controlled by promoter-enhancer sequences of a humancytomegalovirus early gene (CMV promoter), which allowed a high level ofexpression. There was an untranslated region at the 3′ end of luc,originating from the virus SV40, which contained the polyadenylationsignal (poly(A)+). After intermediate cloning, which allowed the numberof available restriction sites to be increased, the “CMVpromoter-luc-poly(A)+” cassette was introduced into the minimal vectorori γ-cer-sup Phe (pXL2760) in place of the Km^(R) marker. The resultingplasmid has been named pXL2774. FIG. 6 shows the various cloning steps.The ligation mixtures were transformed into XAC-1pir116 byelectroporation. Incubation allowing the bacteria to express selectionmarkers was carried out in rich medium (SOC medium); it was thusnecessary to wash the cells twice with M9 medium before plating out.This made it possible to remove the residual medium, which would haveresulted in culture background noise on minimal medium.

The medium chosen to plate out the electroporated cells was M9 minimalmedium, which makes it possible to select bacteria expressing asuppressor tRNA and thus the presence of our plasmids. The addition ofX-Gal made it possible, by means of the blue colouration, to visualizethe expression of the suppressor tRNA. The dishes were analysed afterabout 20 hours at 37° C. The absence of colonies on the DNA-free controlassures us that the selection was correct, even with dense seedings. Allthe clones examined by restriction (8) do indeed carry a plasmid,corresponding to the expected profile. The plasmid thus constructed,pXL2774, was prepared from a clone cultured in one liter of liquid M9medium (about 18 hours at 37° C.), by a technique involving, inter alia,an ion-exchange step (Promega kit, MegaPreps). The amount of DNAcollected was 2 mg.

2) Analysis of the Reporter Vector pXL2774 Transfected into MammalianCells.

The capacity of pXL2774 to transfect eukaryotic cells and to allow theexpression of luciferase was evaluated by transfection into NIH 3T3mouse fibroblasts. The vector chosen as reference was the plasmidpXL2622 (this is the plasmid pGL2 from Promega whose SV40 promoter hasbeen replaced by the CMV promoter), which carries the same luciferaseexpression cassette as pXL2774, but on a different replicon. This is a6.2 kb ColE1 derivative which carries the ampicillin-resistance gene.This plasmid served as a control. The luciferase activities (expressedas RLU, or relative luminescence units) are indicated in Table 3.

The best results were obtained with a “lipofectant charges/DNA charges”ratio of 6; under these conditions, pXL2622 and 2774 appear to beequivalent.

TABLE 3 pXL2622 pXL2774 RLU/μg of Coefficient RLU/μg of CoefficientCharge proteins and of variation proteins and of variation ratios perwell Average (%) per well Average (%) 0 0.0 not 0.0 not 0.0 detectable0.0 detectable 0.0 0.0 3 9.9 10⁶ 7.6 10⁶ 22 3.3 10⁶ 2.9 10⁶ 13 6.2 10⁶2.9 10⁶ 6.6 10⁶ 2.4 10⁶ 6 1.2 10⁷ 1.5 10⁷ 19 9.4 10⁶ 1.0 10⁷ 7 1.5 10⁷9.9 10⁶ 1.9 10⁷ 1.1 10⁷ 9 9.5 10⁶ 1.0 10⁷ 26 1.1 10⁷ 6.4 10⁶ 13 7.5 10⁶8.3 10⁶ 1.4 10⁷ 8.5 10⁶

EXAMPLE 4 Verification of the Suicide Vector Nature in E. coli of thepCOR Plasmids

The non-replicative nature of the pCOR-type plasmids derived from R6Kwas verified by an electroporation experiment in JM109 E. coli(Yanisch-Perron et al., 1985) of plasmids pUC4K (ori ColEI-KmR, (Vieiraand Messing, 1982)) and pXL2730 (ori gamma from R6K-KmR, see Example 2).The electroporator used was the Biorad Gene Pulser and theelectrocompetent JM109 cells were prepared and used according to themanufacturer's procedure (Bacterial electro-transformation and pulsecontroller instruction manual. catalog number 165-2098).

The electrotransformed cells were plated out on LB medium supplementedwith kanamycin (50 mg/l) and incubated overnight at 37° C. The resultsobtained are presented below.

Results Efficacy (number of Amount transformed Number of transformants/Plasmid (ng) transformants ng of plasmid) pUC4K 0.01 >>2000 >2105pXL2730 5 0 0

These results show that there was a minimum of 5 logs of differencebetween the efficacy of transformation of a ColEI derivative (pUC4K) andthat of an R6K derivative (pXL2730) in a strain which does not expressthe pir gene. In a pir+ strain such as XAC-1pir116, theelectrotransformation efficacy of R6K-derived plasmids conventionallyreaches or exceeds the 108 transformants/μg of plasmid.

EXAMPLE 5 Production of plasmid DNA by high-density culturing of the E.coli strain XAC-1pir116 (pXL2774): fermentation process

5.1 Strains:

Production in XAC-1pir116 E. coli (Example 1) of a minimal plasmid,pXL2774; this plasmid comprises the following elements: oriR6K-cer-tRNAamsupPhe and an expression cassette of the luc reporter geneunder the control of the CMV promoter (Example 3). A high-productivityprocess for the production of plasmids of this type was developed.

5.2 Culturing Media and Conditions:

a) Growth Medium:

Composition of the medium defined used for the inoculum cultures (g/l):Na₂HPO₄ 6, KH₂PO₄ 3, NaCl 0.5, NH₄Cl 1, NH₄H₂PO₄ 3, glucose 5,MgSO₄.7H₂0 0.24, CaCl₂.2H₂O 0.015, thiamine HCl 0.010

Composition of the complex medium used for the fed-batch cultures (g/l):KH₂PO₄ 8, K₂HPO₄ 6.3, Na₂HPO₄ 1.7, (NH₄)₂SO₄ 0.74, NH₄C10.12, yeastextract 3, glucose 2, MgSO₄.7H₂O 2.4 g/l, CaCl₂.2H₂O 0.015, thiamine0.010, solution of salts (Fe, Mn, Co, Zn, Mo, Cu, B, Al).

Composition of the medium defined for the cultures in fed-batch mediumidentical to the complex medium, but the yeast extract is replaced by2.5 g/l of NH₄Cl.

b) Conditions of Fed-Batch Culturing:

Studies in 2-liter fermenters (Setric France) containing 1 l of mediumwere carried out in order to define the optimum conditions for growingand producing plasmid DNA. The fermenter was inoculated with 80 ml of aninoculum culture arrived at the start of the stationary phase of growth.

During the fermentation, the pH was controlled and adjustedautomatically between 6.9 and 7.0 with 10% (w/v) aqueous ammonia; thetemperature was maintained at 37° C.; the aeration was set at 75 l/h((1.1 vvm) at a pressure of 0.2 bar and the dissolved oxygen wasadjusted to (40% of air saturation by retroaction on the stirring rateand, if necessary, by enrichment with pure oxygen.

All the parameters (pH, temperature, stirring, OD, O₂ and CO₂ in theeffluent gases) were collected and calculated in line via an HP3852interface connected to a Hewlett-Packard 9000.

The base composition of the supply medium was as follows: 50% carbonsource, 0.7% magnesium sulphate, 0.02% thiamine; for the complex medium,yeast extract was added to a concentration preferably of between 5 and10%.

To adapt the culture conditions to 800-liter fermenters, productionsequences composed of two successive inoculum cultures were carried out,on a laboratory scale: inoculum I in an agitated conical flask andinoculum II in a 2-liter fermenter (batch culturing), followed byfed-batch production culturing, in a 7-liter fermenter.

5.3 Results

Various culture conditions were studied in complex medium, in definedmedium, and at various growth rates. In all cases, after initial batchculturing of the bacterial strain and consumption of the carbon source,the supply medium was added to the fermenter by means of a peristalticpump coupled to a pre-programmed addition profile. This profile wasdeduced from previous experiments in which the supply rate had beencontrolled either by the level of dissolved oxygen or by a constantgrowth rate.

Furthermore, in order to extrapolate without difficulty the 2-literfermentation condition to an 800 l fermenter without overoxygenation ofthe medium, the maximum oxygen demand at the end of the culturing wasset at 2.5-3 mM/min. For this, the growth rate of the microorganism wasreduced, if necessary, by varying the supply rate of the complementarycharge.

As seen in Table 4, very good results were obtained both in complexmedium and in defined medium, both on the laboratory scale and on the800-liter fermenter scale; furthermore, the plasmid DNA growth andproduction kinetics are entirely comparable (cf. FIGS. 6 and 7).

TABLE 4 Defined Complex medium medium 2 or 7 l 800 l 2 l fermenterfermenter fermenter Duration of 40 39 48 fermentation (hours) μh-l 0.1300.132 0.124 OD (600 nm) 114 100 94 X g/l 44 37 30 Plasmid DNA 115 100100 (mg/l medium) Plasmid DNA 2.6 2.7 3.3 (mg/gX) X = Biomass (weight ofdry cells)

From the overall results it emerges that:

-   -   changing the scale of the fermenter from 2 liters to 800 liters        can be carried out without any problem,    -   the oxygen consumed is strongly correlated to the biomass        produced (1.08 g O₂/g of biomass produced),    -   the plasmid was stable for at least 50 generations without        selection pressure,    -   a high biomass, greater than 40 g of dry cells/liter, can be        obtained in complex medium,    -   the plasmid DNA production reaches 100 mg of supercoiled DNA/I        of medium,    -   there was very good correlation between the DNA production and        the biomass: the production can be estimated to (1 mg of plasmid        DNA/OD unit, or alternatively (2.7 mg of plasmid DNA/g of        biomass, irrespective of the duration of fermentation,    -   the use of a defined medium also makes it possible to achieve a        high biomass (30 g of dry cells/l) and high plasmid DNA        production (100 mg/l), without any loss of productivity.

EXAMPLE 6 Transfer of pXL2774 into Animal Cells, In Vitro and In Vivo

6.1 In Vitro Transfer of pXL2774 into Animal Cells

The capacity of the minimal plasmid pXL2774 to transfect various celllines was tested in vitro, on cells of both human origin and murineorigin. The pXL2784 plasmid was used as control. It contains the sameeukaryotic expression cassette (CMV promoter-luciferase-polyA) aspXL2774, but this is a 6.4 kb ColE1 derivative which comprises the genefor imparting kanamycin resistance in E. coli.

The cells tested are the following:

Atcc ref./ Cells Type literature ref. 3LL Mouse pulmonary carcinoma NIH3T3 Mouse embryo fibroblasts CCL92 293 Human embryo renal cellstransformed CRL1573 with type-5 adenovirus HeLa Human carcinoma from theneck of the CCL2 womb Caco-2 Human colon adenocarcinoma HTB37 H460 Humanlung carcinoma with no small HTB177 cells ECV 304 Human umbilical cordendothelial cells Takahashi et al., 1990

The transfection conditions were as follows:

D-1: Inoculation of the cells at a density of 100,000 cells per 2 cmwell (24-well plate) in DMEM medium (Dulbecco's modified Eagle Medium)supplemented with 10% fetal calf serum (FCS).

D-3: Transfection of the cells, by 10 μl of a transfection solutioncontaining: 0.5 μg of DNA, 150 mM NaCl, 5% glucose and 3 nmol of RPR120535 lipofectant per μg of DNA, in 250 μl of culture medium, which was orwas not supplemented with 10% FCS. After incubation for 2 hours, themedium was replaced by 500 μl of DMEM medium supplemented with 10% FCS.

D-4: Renewal of the culture medium

D-5: Washing of the cells with PBS, followed by lysis with 100 μl ofPromega lysis buffer (Promega Cell Lysis Buffer E153 A). Assay of theluciferase activity was carried out in a Lumat LB 9501 luminometer(Berthold) on 10 μl of lysate, with a 10-second duration of integration.The reactant used was that from Promega (Promega Luciferase AssaySubstrate). The results, collated in the following tables 5-8, areexpressed in RLU (Relative Lights Units) for 10 μl of lysate (average ofmeasurement on 4 wells). The coefficients of variation (CV) are alsogiven.

The results of transfections in the absence of serum are presentedbelow.

CELL TYPES NIH 3T3 3LL 293 pXL2774 37 763 380 559 270 1 884 200 RLU 1625  73 CV pXL2784 113 764 1 723 546 RLU 24 101 CV CELL TYPES HeLa CaCo2H460 ECV304 pXL2774 11 000 000 1 108 422 1 459 501 36 450 15 14  5 23pXL2784 557 930 93 610 7 563 168 795 87 40 11 40

The results of transfections in the presence of serum (10%) arepresented below:

CELL TYPES NIH 3T3 3LL 293 pXL2774 50 612 590 566 377 992 500 12 18 59PXL2784 12 693 780 436 704 2 300 000 38 12 47 CELL TYPES HeLa H460ECV304 pXL2774 9 490 000 857 385 18 021 25 16 30 PXL2784 1 508 480 433023 32 074 23 27 47

These results reveal the capacity of pXL2774 to transfect effectively,in vitro, various cell types of both murine and human origin. Theexpression of the luc reporter gene made it possible to show that itstransfection efficacy is at least as good as that of a “standard”plasmid, derived from ColE1, which carries the same expression cassetteof luciferase.

6.2. In Vivo Transfer in Animals (Mice), of pXL2774

a) Model 1: Naked DNA in Mouse Cranial Tibial Muscle

Naked plasmid DNA, dissolved in “5% glucose, 150 mM NaCl” was injectedinto the cranial tibial muscle of OF 1 mice. The muscles were removed 7days after injection, chopped up, homogenized in 750 μl of lysis buffer(Promega Cell Lysis Buffer E153A) and then centrifuged at 20,000×g for10 minutes.

Assay of the luciferase activity was carried out on 10 μl of supernatantafter addition of 50 μl of reagent (Promega Luciferase Assay Substrate).The reading was carried out on a Lumat LB9501 luminometer (Berthold)with a 10-second duration of integration.

The results are presented in the table below.

Plasmid pXL2784 pXL2774 pXL2784 pXL2774 Number of 8 8 10 10 muscles:Volume injected 30 30 33 33 (il): μg of 19 13.5 10 6.25 DNA/muscle RLU(for 10 μl) Average 80 922 471 733 35329 30569 Standard deviation 104573 402 602 37041 35774

These results show that a conditional replication plasmid such aspXL2774 was indeed capable of transfecting mouse muscle cells in vivoand of doing so with comparable, or even superior, efficacy to that of a“standard” plasmid derived from ColE1, which carries the same expressioncassette of the luciferase gene.

b) Model 2: 3T3 HER2 tumour model

The model is as follows:

-   -   Swiss/nude adult female type mice    -   Experimental tumours induced after injection of 107 3T3 HER2        cells subcutaneously into the flank.    -   The transfection mixture was injected 7 days after injection of        the cells.    -   Solutions injected: The DNA was first dissolved in the buffer.        After addition of all the products, the mixture contained,        besides the DNA, NaCl (150 mM) and 5% D-glucose in water or 5 mM        HEPES buffer.    -   Two days after the injection, the tumour tissue was removed,        weighed and then chopped up and homogenized in 750 μl of lysis        buffer (Promega Cell Lysis Buffer E153 A). After centrifugation        (20,000×g for 10 minutes), 10 μl of supernatant was removed and        luciferase activity was evaluated. This activity was determined        by measuring the total light emission obtained after mixing with        50 μl of reagent (Promega Luciferase Assay Substrate) in a Lumat        LB 9501 luminometer (Berthold) with a 10-second duration of        integration.

The resulting activity was expressed in RLU (Relative Light Units)estimated in the entire tumour lysis supernatant.

Results Plasmid Buffer [DNA] RLU/tumour results H20 or final in standardHEPES reference μg/tumour inj. sol. average deviation +/n HEPES pXL278410 0.5 μg/μl   744 150   682 434 6/6 pXL2774 10 0.5 μg/μl 1 016 380 1322 500 5/6 H2O pXL2784 24 0.6 μg/μl 2 906 073 1 745 857 8/8 pXL277416.8 0.4 μg/μl 4 292 043 4 995 187 6/6 H2O pXL2784 7.5 0.3 μg/μl   702554   552 207 6/7 pXL2774 5 0.2 μg/μl 3 413 430 4 000 875 6/6

These results show that a conditional replication plasmid, such aspXL2774, was indeed capable of transfecting mouse tumour cells in vivoand of doing so with an efficacy at least comparable to that of a“standard” plasmid, derived from ColE1, which carries the sameexpression cassette of the luciferase gene.

These various experiments demonstrate that the conditional replicationplasmids, and more particularly pXL2774, did indeed have animal celltransfection characteristics that are essential for use in gene therapy.More precisely, the following were shown:

-   -   1) the capacity of pXL2774 to transfect efficiently, in vitro,        various cell types of human or murine origin;    -   2) the capacity of pXL2774 to transfect, in vivo, mouse muscle;    -   3) the capacity of pXL2774 to transfect, in vivo, tumour cells        implanted into mice.

The electrotransformation, fermentation and transfection experimentsthus made it possible to validate conditional replication plasmids asvectors which can be used in gene therapy by showing:

i) that they did not replicate detectably in an E. coli strain that doesnot express the pir gene (conditional origin of replication)

ii) that they could be produced on a scale compatible with industrialproduction, in a defined medium that does not contain antibiotics;

iii) that these plasmids could transfect, in vitro and especially invivo, mammalian cells.

EXAMPLE 7 In Vitro Production of Recombinant Proteins

7.1 Construction of the Expression Vector

To show the feasibility of such an approach, we constructed anexpression vector according to the criteria described above (Examples 2and 3). This vector, pXL3056, contains:

1) the bacterial part which comprises the conditional origin ofreplication (ori gamma), the cer fragment of ColE1, and the gene whichensures selection in bacteria (sup)

2) the expression cassette, based on the system described by Studier(Studier et al., 1990), comprising the promoter of gene 10 ofbacteriophage T7, the lacO operator, the gene coding for aFGF 154(acidic Fibroblast Growth factor, form containing 154 amino acids) (Jayeet al., 1986), and the TF terminator of bacteriophage T7. Thisexpression cassette is identical to the one present on the pXL2434plasmid, which is described in application WO 96/08572.

The construction of pXL3056 is presented in FIG. 8. The EcoRI-BglIIfragment of pXL2434 (1.1 kb) containing the aFGF expression cassette wascloned in the pXL2979 conditional replication vector (1.1 kb purifiedfragment) at the BglII and EcoRI sites to generate pXL3056.

pXL2979 results from the ligation of 3 fragments: i) AccI-XbaI fragmentof pXL2730 (0.8 kb, which provides ori gamma and cer), ii) NarI-SalIfragment of pXL2755 (0.18 kb, which provides the sup Phe gene), iii)SalI-SpeI fragment of pXL2660 (1.5 kb, which provides the kanamycinresistance gene).

pXL2660 results from the cloning of the 1.2 kb PstI fragment of pUC4K(Vieira and Messing, 1982) in pMTL22 (Chambers et al., 1988) linearizedwith PstI.

7.2 Production of the Expression Strain

Plasmid pXL3056 was introduced by transformation into the XAC-1pir116strain. The resulting strain was then transformed by the plasmidPT7pol23 (Mertens et al., 1995), at 30° C. In order to express the geneof interest under control of the T7 promoter, the bacterium must containin its genome, on a plasmid, or a bacteriophage, a cassette allowingexpression of the RNA polymerase of bacteriophage T7. In the exampledescribed, we used the plasmid pT7pol23, which is compatible with R6Kderivatives such as pXL3056, and which allows the temperature-inducibleexpression of bacteriophage T7 RNA polymerase. However, it can also beenvisaged to lysogenize the XAC-1pir116 strain with lambda DE3 (Studieret al., 1990) to conserve only one plasmid and to induce the productionof T7 RNA polymerase by IPTG rather than by temperature.

7.3 Expression of aFGF

The XAC-1pir116 strain (pXL3056+PT7pol23) was cultured at 30° C. in M9minimum medium supplemented with 0.2% of casamino acids (DIFCO) andkanamycin (25 μg/ml), up to an optical density at 600 nm of 0.6-1.0.Half of the culture was then placed at 42° C. (induction of the T7 RNApolymerase), while the other half remained at 30° C. (negative control).The same experiment was carried out with the XAC-1pir116 (pXL3056+pUC4K)strain which constitutes a control for the expression of aFGF in theabsence of T7 RNA polymerase.

The results obtained are presented in FIG. 9. They show that theproduction of aFGF was comparable or superior to that observed withBL21(DE3)(pXL2434) (WO 96/08572), which clearly shows the potential ofconditional replication plasmids for the expression of recombinantproteins in vitro, especially in E. coli.

EXAMPLE 8 Construction of a pCOR Vector which Expresses a Wild-Type orHybrid p53 Protein or the FGF1 Human Protein

This example describes the construction of conditional replicationvectors according to the invention containing a nucleic acid coding fora p53 protein. These vectors can be used to restore a p53-type activityin deficient (mutated, deleted) cells such as, in particular, tumourcells.

The eukaryotic expression cassette contains the following elements:

1) CMV “immediate early” promoter (positions −522 to +72) followed bythe leader sequence of the thymidine kinase gene of type I herpessimplex virus (position −60 to +1 of the gene, with reference to thesequence in the article by McKnight, S.†L. (1980) Nucleic Acids Res.8:5949-5964);

2a) a nucleic acid which codes for wild-type p53 protein or for a p53variant, as described in application PCT/FR 96/01111 (V325K variant=V325with a Kozak sequence with ATG);

2b) a nucleic acid which codes for the human FGFa or FGF-1 as describedin Jaye M. (Sciences 1986; 233(4763):451, U.S. Pat. No. 4,686,113, andEuropean Patent No: 259 475;

c) a nucleic acid which codes for a fusion gene between human fibroblastinterferon secretion signal (Taniguchi et al.) and the naturallyoccurring truncated form of human FGF-1 from amino acid 21 to 154 asdescribed by Jaye et al., and U.S. Pat. No. 5,849,538.

3) the polyA polyadenylation sequence of SV40.

These elements were placed in the form of a fragment AscI-XbaI on thepCOR vector pXL2988 between the sites BssHII and SpeI. pXL2988 isidentical to pXL2979 (Example 7.1.) apart from the presence of anadditional element, a sequence capable of forming a DNA triple helixcomposed of 17 copies of the trinucleotide GAA, placed alongside thegamma origin of replication.

The resulting plasmids were named pXL3029, pXL3030, pXL3179 or NV1FGF(FIG. 10).

The nucleic acid sequence of pXL3179 is set forth in SEQ ID NO: 40:CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGTCTAGAGCCTTCGAAGCTTGCCATGACCAACAAGTGTCTCCTCCAAATTGCTCTCCTGTTGTGCTTCTCCACTACAGCTCTTTCCATGAATTACAAGAAGCCCAAACTCCTCTACTGTAGCAACGGGGGCCACTTCCTGAGGATCCTTCCGGATGGCACAGTGGATGGGACAAGGGACAGGAGCGACCAGCACATTCAGCTGCAGCTCAGTGCGGAAAGCGTGGGGGAGGTGTATATAAAGAGTACCGAGACTGGCCAGTACTTGGCCATGGACACCGACGGGCTTTTATACGGCTCACAGACACCAAATGAGGAATGTTTGTTCCTGGAAAGGCTGGAGGAGAACCATTACAACACCTATATATCCAAGAAGCATGCAGAGAAGAATTGGTTTGTTGGCCTCAAGAAGAATGGGAGCTGCAAACGCGGTCCTCGGACTCACTATGGCCAGAAAGCAATCTTGTTTCTCCCCCTGCCAGTCTCTTCTGATTAACTCGAGCATGCATCTAGGCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTAGCCCGGGCGCGCAGATCTGTCATGATGATCATTGCAATTGGATCCATATATAGGGCCCGGGTTATAATTACCTCAGGTCGACGCGTCTGCAGAAGCTTAAAAAAAATCCTTAGCTTTCGCTAAGGATCTGCAGTGCCCGGACTCGGAATCGAACCAAGGACACGGGGATTTAGAATCCCCTGCTCTACCGACTGAGCTATCCGGGCGCGTTACAAGTATTACACAAAGTTTTTTATGTTGAGAATATTTTTTTGATGGGGCGACCTGCAGGTCGGGGCACAACTCAATTTGCGGGTACTGATTACCGCAGCAAAGACCTTACCCCGAAAAAATCCAGGCTGCTGGCTGACACGATTTCTGCGGTTTATCTCGATGGCTACGAGGGCAGACAGTAAGTGGATTTACCATAATCCCTTAATTGTACGCACCGCTAAAACGCGTTCAGCGCGATCACGGCAGCAGACAGGTAAAAATGGCAACAAACCACCCGAAAAACTGCCGCGATCGCGCCTGATAAATTTTAACCGTATGAATACCTATGCAACCAGAGGGTACAGGCCACATTACCCCCACTTAATCCACTGAAGCTGCCATTTTTCATGGTTTCACCATCCCAGCGAAGGGCCATCCAGCGTGCGTTCCTGTATTTCCGACGGATCCGGCCACGATGCGTCCGGCGTAGAGGATCTGAAGATCAGCAGTTCAACCTGTTGATAGTACGTACTAAGCTCTCATGTTTCACGTACTAAGCTCTCATGTTTAACGTACTAAGCTCTCATGTTTAACGAACTAAACCCTCATGGCTAACGTACTAAGCTCTCATGGCTAACGTACTAAGCTCTCATGTTTCACGTACTAAGCTCTCATGTTTGAACAATAAAATTAATATAAATCAGCAACTTAAATAGCCTCTAAGGTTTTAAGTTTTATAAGAAAAAAAAGAATATATAAGGCTTTTAAAGCTTTTAAGGTTTAACGGTTGTGGACAACAAGCCAGGGATGTAACGCACTGAGAAGCCCTTAGAGCCTCTCAAAGCAATTTTGAGTGACACAGGAACACTTAACGGCTGACA TGGGAATTCTAGTAAATGCC

Plasmid pXL3179, which is also referred to as NV1FGF, was deposited inhost strain TEX1 under the terms of the Budapest Treaty with theCollection Nationale de Cultures de Microorganismes (CNCM), InstitutPasteur, 25, rue du Docteur Roux, F-75724, Paris Cedex 15 France, onFeb. 2, 2006, under accession number CNCM I-3569.

The functionality of these constructions was verified in vitro onp53-SAOS2 cells in culture by measuring the transcriptional-activatoractivity of p53 or p53superWT, or by measuring the secretion of FGF 1for example by ELISA experiments which is well known in the art.

EXAMPLE 9 Construction of TEX 1 (XAC1 pir116, endA⁻, traD⁻)

The E. coli XAC-1pir116 contains an F′ episome, a circular DNA moleculeof approximately 100 kb, that carries proB+lacI₃₇₃lacZ_(u118am). Manymale E. coli laboratory strains carry a traD36 mutation on theirepisome, but no mutation affecting F′ transfer ability has beendescribed for XAC-1. The gene traD is at the 5′ end of one of the tra(transfer) operons and encodes a membrane protein directly involved inDNA transfer and DNA metabolism (Frost et al., BBRC, 1994, 58:162-210).A 2 kb central fragment from traD, comprising 92% of the gene, wasreplaced with the 2 kb omega element (Genbank accession number M60473)from pHP45Q (Prentki and Krisch, 1984, Gene, 29:303-313) by homologousrecombination in XAC-1pir116 endA⁻. The omega element contains the aadAantibiotic resistance gene flanked by short inverted repeats. The geneaadA encodes aminoglycoside-3 adenyltransferase and confers resistanceto streptomycin and spectinomycin (“Sp^(R)”). The omega fragment wasused because it prematurely terminates RNA and protein synthesis leadingto the inactivation of the whole traD operon. This new pCOR strainXAC-1pir116 end-traD::SpR was designated TEX1. Transfer of any residentplasmids, either pCOR or pUC, was undetectable when the donor was TEX1.

The new pCOR host strain TEX1 was assessed in fermentation experiments.Complex media containing yeast extract were used for fed-batchfermentation with XAC-1pir116. pCOR stability (more than 50 generations)makes it possible to use a non-selective media. Under these conditions,XAC-1pir116 produced more than 40 g/l dry cell weight and 100 mg/l ofpCOR pXL2774 were obtained from 2-liter fermenters. pCOR copy number wasestimated at 400-500 copies per cell and the rate of plasmid DNAsynthesis was constant throughout fermentation. These results wereextrapolated to an 800-liter fermenter suitable for manufacturing. Thefermentation was also performed in the absence of yeast extract or anyraw material from animal origin. Similar results (30 g/l dry cell weightand 100 mg/l of plasmid DNA) were obtained using a defined medium in2-liter cultures with no loss of productivity.

EXAMPLE 10 Construction of XAC-1pir116pir42 Host Strains by HomologousRecombination

1) Construction of a Suicide Vector Carrying the Cassette“KmR-uidA:pir116:pir42”

The Km^(R)-uidA::pir116 cassette from M13wm33 as described in Example 1(Metcalf W. et al. Gene, 1994, 138(1-2): p. 1-7), was modified bysite-directed mutagenesis using PCR (QuickChange site-directedmutagenesis kit, Stratagene, La Jolla, Calif.) to introduce the pir42mutation into the pir116 gene. The different cloning/mutagenesis stepsare described in FIG. 13

The oligonucleotides used for mutagenesis contained the pir42 mutationalong with a silent mutation that created a ClaI site to easily indicatethe processing of pir42 by restriction analysis when needed.

The sense and antisense oligonucleotides used are as follows:

Sense oligonucleotide number 11076 (SEQ ID NO: 7) 5′-G TAT ATG GCG CTTGCT CTC ATC GAT AGC AAA GAA                          pir42 ClaI CC-3′Antisense oligonucleotide number 11077 (SEQ ID NO: 8) 5′-GG TTC TTT GCTATC GAT GAG AGC AAG CGC CAT ATA                     ClaI pir42 C-3′

The technique used to replace pir116 by pir116pir42 in the genome of E.coli pCOR host TEX1 was based on that of Blum et al. (J. Bacteriol.1989, 171, pp 538-46). The recombinant bacteriophage pXL3723 shown inFIG. 13 is a suicide vector in all non-suppressor E. coli strains,because it has a non-sense mutation in gene II encoding M13 nickase thatprevents viral genome replication.

Double recombination was performed as described for the construction ofXAC-1pir116 (Example 1, point 2). Clones that had undergone doublehomologous recombination events were screened by PCR to test for thepresence of the pir42 mutation in the genome of TEX1. Genomic DNAisolated from double recombination candidates was used as a template forPCR. Secondly, sequencing was done on each unique amplified fragment,all of which were of the expected size. The PCR fragments are shown inFIG. 14.

The PCR primers were the following:

Primer 11088 (SEQ ID NO: 9): 5′-GAGATCGCTGATGGTATCGG-3′ Primer 11089(SEQ ID NO: 10): 5′-TCTACACCACGCCGAACACC-3′

This analysis showed that one out of the six double recombinants testedhad undergone the allele exchange. This new strain, named TEX1pir42, wasfurther evaluated for its ability to replicate pCOR plasmids compared tothe parental strain TEX1.

2) Evaluation of TEX1pir42

pCOR plasmids were transformed in parallel into TEX1 and TEX1pir42 andgrown overnight in 2 ml of selective M9 medium. Then, the plasmid DNAwas extracted with the Wizard SV plus minipreps kit (Promega) toevaluate the relative plasmid copy number and topology of the pCORplasmids in both strains.

A 2-fold increase in copy number was obtained reproducibly in TEX1pir42transformed with the pCOR plasmid pXL3516 (2.56 kb). To furthercharacterize TEX1pir42, the copy number and topology of pCOR plasmidssuch as pXL3179 and pXL2774 were evaluated by agarose gelelectrophoresis analysis after small scale purification of plasmid DNA(4 to 6 clones/strain). Copy number was evaluated on plasmids linearizedwith EcoRI restriction enzyme. A topology test was run on non-digestedplasmids, in the absence of ethidium bromide. The resulting agarose gelis displayed in FIG. 15, and clearly shows a higher plasmid copy numberwhen the plasmid pXL3179 was produced in TEX1pir42, than when producedin TEX1 strain. FIG. 15 also displays the topology of the plasmidpXL3179, and shows that an increase in plasmid copy number, which wereessentially in the form of monomers, with few plasmids in the form ofmultimers. The results obtained with these pCOR plasmids are alsosummarized in Table 5. Relative copy number was calculated in comparisonwith the same plasmid in TEX1. A 2-3 fold increase in plasmid copynumber was observed with plasmids pXL3179 and 2774 produced inTEX1pir42.

TABLE 5 Replication and copy number of pCOR plasmids produced inTEX1pir42 RELATIVE COPY PLASMIDS SIZE (kb) NUMBER* pXL3179 2.4 x3pXL2774 4.5 x2 *copy number was compared to the same plasmid in TEX1.

EXAMPLE 11 Comparative Experiments: Construction of TEX1cop21(XAC-1endA-traD-pir116cop21)

1) Construction of TEX1cop21

The TEX1cop21 strain was constructed similarly as that described inExample 10 for TEX1pir42. The following oligonucleotides used tointroduced cop21 into the pir116 gene by directed mutagenesis were asfollows:

Sense oligonucleotides: 11153 (SEQ ID NO: 11) 5′-CG CAA TTG TTA ACGTCC AGC TTA CGC TTA AGT AGC                       cop21 C-3′ Antisenseoligonucleotide: 11154 (SEQ ID NO: 12) 5′-G GCT ACT TAA GCG TAA GCT GGACGT TAA CAA TTG CG-3′

The cop21 mutation was introduced as a TCC serine codon instead of theTCA serine codon to eliminate a HindIII restriction site close to themutation.

The template used for directed mutagenesis was pXL3395 (see FIG. 13).The resultant plasmid named pXL3432 was used to construct the suicideM13 vector in a similar way as to what is shown for pir42 in FIG. 13.The suicide vector pXL3749 is shown in FIG. 16.

The E. coli clones obtained after homologous recombination with pXL3749were screened by PCR and subsequent restriction with HindIII andsequencing to monitor the cop21 and pir116 mutations. One clone out ofthe six double recombinants tested had undergone the gene replacement.The resulting strain was named TEX1cop21.

2) Evaluation of TEX1cop21

TEX1cop21 was transformed by various pCOR plasmids, including pXL2979, a2.5 kb Km^(R) pCOR vector (See Example 7.1), and assayed for increasedcopy number by gel electrophoresis. Such an experiment with pXL2979 isshown in FIG. 17. Plasmid DNA from four independent clones for eachstrain prepared with Promega miniprep kit was linearized with EcoRI,electrophoresed on agarose gel and then stained with ethidium bromide.Each sample represented a similar amount of bacteria, as measured byoptical density at 600 nm. The agarose gel electrophoresis obtained forthe pCOR plasmid pXL2979 produced in E. coli TEX1cop21, XAC1pir, andTEX1 is displayed in FIG. 17. It clearly shows there was no increase inplasmid copy number when the plasmids are produced in the TEX1cop21strain, as compared with TEX 1.

EXAMPLE 12 Construction of TEX2pir42 (XAC-1pir116 pir42 recA)

Firstly, a recA-derivative of TEX1 was constructed. The pir42 mutationwas then introduced into the resulting strain named TEX2 to generateTEX2pir42.

1) Construction of E. coli TEX2, a recA-Derivative of TEX1

A deleted recA gene containing 3 translation stop codons (one in eachframe) at its 5′ end was obtained by PCR. This deleted recA gene wasintroduced by gene replacement (Blum et al., J. Bacteriol., 1989, 171,pp. 538-46) into the TEX1 genome. The construction of the suicide vectorfor homologous recombination is shown in FIG. 18.

PCR primers used for the amplification of recA fragments are shown inthe following Table 6:

TABLE 6 primers DNA sequences seq 5′CCCTCTAGATCGATAGCCATTTTTACTCCTG 3′10930 SEQ ID NO: 13 seq 5′CGGGATCCTGATTATGCCGTGTCTATTAG 3′ 10931 SEQ IDNO: 14 seq 5′ CCCAAGCTTCTTCGTTAGTTTCTGCTACGCCTTCGC 3′ 10932 SEQ ID NO:15 seq 5′GGTCTAGAACGTGAAAGTGGTGAAGAACAAAATCG 3′ 10933 SEQ ID NO: 16

Restriction sites added to the recA sequence are underlined.

To maintain the RecA+ phenotype necessary for homologous recombinationto occur, the recA function was provided to E. coli TEX1 with a plasmidcontaining a heterologous recA gene that can complement E. coli recAmutants, such as for example the recA gene of the bacteriumAgrobacterium radiobacter, and an antibiotic gene resistance, such asthe ampicillin resistance gene. After gene replacement, the plasmid waseliminated from the recombinant strain by culture-dilution innon-selective medium (LB). The absence of the plasmid was screened forthe loss of antibiotic resistance.

The resulting strain was named TEX2. Gene replacement was monitored byPCR in FIG. 19. PCR Primers are described in the following Table 7.

TABLE 7 Primers 11355-11354 Primers 11355-11354 Wild type recA 1117 bp1089 bp Deleted recA  404 bp  376 bp

The first primer was based on the sequence of the recA gene. The secondone was based on a sequence close to but outside the homology regionpresent in the suicide vector pXL3457 (immediately 5′ or 3′ of recA) toensure that amplification can only occur on a genomic fragment. Thesequence of both oligonucleotides was chosen according to the sequenceof E. coli, which comprises the recA locus (Genbank ECAE000354).

The PCR fragments obtained from a recA-deleted strain were shorter ascompared to those obtained with a wild-type strain, as presented in thefollowing Table 8.

TABLE 8 PCR primers for amplication of recA primers 5′->3′ sequence seq11352-SEQ ID NO: 17 GCGACCCTTGTGTATCAAAC seq 11353-SEQ ID NO: 18GGTATTACCCGGCATGACAG seq 11355-SEQ ID NO: 19 GTGGTGGAAATGGCGATAGG seq11354-SEQ ID NO: 20 GCGATTTTGTTCTTCACCAC

The PCR profile obtained was as expected and demonstrated the presenceof a truncated recA gene in the genome of TEX2. The recA-phenotype(sensitivity to UV light), as well as phenotypic characteristics ofTEX2, were checked. Phenotypic characteristics of TEX2 were the same asthose of TEX1 strain, i.e., ara-, Rif^(R), Nal^(R), Sp^(R), UidA-, Arg-,Km^(S) Amp^(S)), as expected.

B) Construction of E. coli TEX2pir42

A TEX2pir42 strain was constructed by double homologous recombination,according to the strategy described in Example 10, with the exceptionthat recombination in TEX2 was carried out in presence of a plasmidcarrying a heterologous recA gene capable of complementing the E. colirecA mutants, in order to maintain a reca+ phenotype required forhomologous recombination.

Gene replacement was monitored by restriction analysis of the PCRproduct digested with ClaI (see FIG. 14). Gene replacement had occurredin two out of the four-studied double recombinant clones.

C) Evaluation of E. coli TEX2pir42

1) Evaluation at Lab Scale Plasmid Production:

TEX2pir42 was transformed by the pCOR plasmid pXL3179 (2.4 kb).Production of pXL3179 in TEX2pir42 was intensively studied at the labscale, in terms of reproducibility of the improvement of plasmid copynumber, conditions of culture, as well as stability (number ofgenerations). All the studies consistently showed a 2 to 5-fold increaseof plasmid copy number as compared to production of pXL3179 in TEX1under the same conditions. Plasmid copy number was assessed further tothe production of pXL3179 in TEX2pir42, and TEX1pir42 and TEX1 ascomparative experiments. In this experiment, plasmids were extractedfrom identical bacterial biomass, based on the OD at 600 nm, andanalyzed by agarose gel electrophoresis. The gel was stained withethidium bromide after electrophoresis. The agarose gel electrophoresis,which is displayed in FIG. 20, clearly shows that plasmids are producedin TEX2pir42 at high copy number, and advantageously shows that plasmidmultimers are reduced when produced in TEX2pir42 instead of TEX1pir42.

2) Evaluation in Fermenters:

These results were confirmed at a larger scale in 7-liter fermenters, asdescribed below.

a) Composition of Fermentation Media

The composition of the medium used for inoculum cultures was: Na₂HPO₄ 6g/l, KH₂PO₄ 3 g/l, NaCl 0.5 g/l, NH₄Cl 1 g/l, NH₄H₂PO₄ 3 g/l, glucose 5g/l, MgSO₄,7H₂0 0.24 g/l, CaCl₂, 2H₂O 0.015 μl, thiamine HCl 0.010 g/l.

The composition of the medium used for fed-batch culture was as follows:KH₂PO₄ 8 g/l, K₂HPO₄ 6.3 g/l, Na₂HPO₄ 1.7 g/l, NH₄Cl 2.5 g/l, glucose 10g/l, MgSO₄,7H₂O 2.6 g/l, thiamine 0.011 g/l, Biospumex36 antifoam 0.1ml/l, salt mix (see table 9) 2.5 ml/l.

TABLE 9 Composition of salt mix Salt mix Solution Final concentration(g/100 ml) in fed-batch medium FeSO₄,7H₂O 1.6 40 CaCl₂,2H₂O 1.6 40MnSO₄,H₂O 0.4 10 CoCl₂,6H₂O 0.16 4 ZnSO₄,7H₂O 0.08 2 MoO₄Na₂,2H₂O 0.0721.8 CuCl₂,2H₂O 0.04 1 H₃BO₃ 0.02 0.5 AlCl₃,6H₂O 0.04 1

The composition of the supply medium was as follows: 50% glucose, 0.7%,magnesium 0.02% thiamine-HCl, 1% Biospumex36 antifoam.

b) Fermentation Parameters

A 7-liter fermenter containing 3 liters of the fed-batch medium wasinoculated with 1.2% of the inoculum culture. Inoculum was prepared asfollows: 250 ml of the inoculum medium in a 2-liter flask was inoculatedwith 0.25 ml of a frozen cell suspension of the E. coli strain TEX2pir42(pXL3179).

Flasks were incubated for 24 hours at 37° C. at 220 rpm. After 24 hours,different parameters were measured: residual glucose: 0 g/l, OD_(600nm)was 2.7 and pH 6.24.

During fermentation, the pH was controlled and adjusted automaticallybetween 6.9 and 7 with NH₃. The temperature was maintained at 37° C. andthe dissolved oxygen adjusted to a 45% pO2 by retroaction on thestirring rate.

After initial batch culturing of the bacterial strain for about 17 hoursand consumption of the carbon source (glucose), the supply medium wasadded. Glucose and acids, such lactate and acetate, were maintained at aconcentration close to 0.

c) Results

Final results are presented in Table 10, as compared to production in a100-liter fermenter with E. coli TEX1 (pXL3179) in optimized conditions.

As for XAC-1pir116, there was no difference between 7-liter and800-liter fermenters in terms of plasmid copy number of pXL3179 producedin TEX1.

Plasmids pXL3179 produced in a 7-liter fermenter using a E. coliTEX2pir42 was compared to the production of pXL3179 in a 100-literfermenter with E. coli TEX 1, in optimized conditions. It wasdemonstrated that as for XAC-1pir116 (See Example 5.3), there is astable plasmid production rate in a 7-liter, 100-liter, or 800-literfermenter in TEX1.

TABLE 10 Characteristics of the fermentation of TEX1 and TEX2pir42strains containing pXL3179. Estimated Duration Concentration copy ofFinal Cell of number fermentation OD Dry DNA (copy/ Reference (h) (600nm) weight (mg/l) bacterium) TEX1 OpGen 43.00 104 33.1 96 616-627(pXL3179) 090 TEX2pir42 Op132 48.47 72 27.1 205 1896-1904 (pXL3179) 8S5

There were 3-fold more copies of plasmid pXL3179 per bacterium inTEX2pir42 as compared to TEX1.

Plasmids corresponding to different fermentation time points wereextracted from identical bacterial biomass, based on the OD at 600 nm,and analyzed by agarose gel electrophoresis. FIG. 21 clearly shows anincrease of the plasmid copy number with the duration of thefermentation. Also, FIG. 21 shows the topology of the pXL3179 plasmidproduced in Op1328S5 TEX2pir42, which was nearly exclusively in amonomeric form.

In conclusion, the E. coli host strain TEX2pir42 according to thepresent invention provided an unexpectedly high plasmid copy numberimprovement of pCOR plasmids, such as pXL3179, of 2 to 5-fold inTEX2pir42 as compared to TEX1, at a lab scale and in fermenters.Furthermore, while the plasmid copy number was greatly improved,plasmids so produced exhibited a monomeric topology, not only atlab-scale but also at a larger scale (7-liter fermenter) compatible withindustrial production.

EXAMPLE 13 New Copy-Up Mutants of pir116 Identified by a NovelFluorescence-Based Screening Method

To increase pCOR plasmid copy number in bacterial host cells, we havemutagenized the pir116 gene, which encodes a copy-up mutated version ofthe pir gene. To date all of the mutations increasing the copy number ofR6K-derived plasmids, such as embodiments of pCOR, have been foundwithin the pir gene.

After random mutagenesis by PCR, mutated pir116 genes were introducedinto a pCOR vector containing the cobA reporter gene, which is describedbelow. After fluorescence based screening, the copy number and topologyof the selected mutant plasmid were evaluated. We obtained threedifferent mutants of pir116 gene that increase plasmid copy number.These novel mutations have not been described previously.

A classical screening method for copy-up mutants is based on antibioticresistance. In this method, the level of resistance of a host bacteriumto an antibiotic is a function of the copy number of an antibioticresistance gene located on a plasmid within the cell. As the copy numberof the plasmid, and therefore the antibiotic resistance gene, increases,the level of antibiotic resistance also increases. This method, however,was not applicable for R6K-derived plasmids in host cells containing thepir116 mutation due to a too high baseline copy number of the plasmid(about 400 copies/cell) in these cells. Accordingly a new screeningmethod based on fluorescence to identify copy-up mutations of the pir116gene was developed.

For this new method, the cobA gene was introduced into a pCOR vector toprovide a simple means of monitoring improvement in plasmid copy number.The cobA gene was obtained from Pseudomonas denitrificans (Crouzet etal., J. Bacteriol, 172:5968-79 (1990)). It encodes uroIIImethyltransferase, an enzyme of the vitamin B12 pathway that adds twomethyl groups to the urogen III molecule. When overexpressed in E. coli,cobA leads to the accumulation of red products that are fluorescentunder near UV light. When exposed to UV, bacterial coloniesoverexpressing this gene appear pink to red. We tested this gene todetermine if it could serve as a reporter gene for plasmid copy numberin the pCOR system.

To evaluate the relationship between plasmid copy number and level offluorescence of transformed bacteria exposed to UV light a controlplasmid (pXL3767) was constructed comprising cobA deleted of its ownpromoter (FIG. 22). This plasmid was transformed into three differenthost strains (XAC1pir, XAC1pir116 and TEX1pir42). These strains wereselected based on previous experiments showing that the average copynumber of a pCOR plasmid in XAC1pir is 1, it is approximately five toten fold higher in TEX1, and 15 to 30 fold higher in TEX1pir42.

Recombinant colonies were streaked on M9 minimal medium and exposed toUV light on a transilluminator as shown in FIG. 22. We observed that thefluorescence intensity of the colonies was positively correlated withthe plasmid copy number, with XAC1pir116 exhibiting more fluorescencethan XACpir, and TEX1pir42 exhibiting more fluorescence than XAC1pir116.

The results shown in FIG. 22 demonstrate that this fluorescence-basedassay method easily discriminates between the tested plasmid copynumbers, especially between the plasmid copy number found in strainsTEX1 and TEX1pir42. That is, the intensity of red fluorescence observedin this assay increases with the plasmid pCOR-cobA copy-number.

Having demonstrated a positive correlation between fluorescence and cobAcopy number, we constructed a plasmid into which mutagenized pir116genes were introduced for screening. Four plasmids with differentcombinations of constitutive modules, as shown in FIG. 23, wereconstructed and tested. One of these plasmids demonstrated asignificantly different level of fluorescence when transformed intopir116 and pir116pir42 isogenic strains. This plasmid, pXL3830, is shownin pertinent part in FIG. 24.

Control plasmids were used during the screening and evaluationexperiments. First, a baseline level control plasmid, pXL3830,containing “wild type” pir116 was used to set a baseline fluorescencelevel. Second, pXL3795, that contains the double mutation pir116-pir42which increases the copy number of the plasmid by 4 to 6 as compared topXL3830, was used as a positive control.

Random mutagenesis was performed on pir116 gene using the Diversify PCRrandom mutagenesis kit (BD Biosciences Clontech, Palo Alto, Calif.,USA). Condition 1, which introduced an average of 2 mutations per 1000base pairs, was used. A preliminary experiment run using “condition 1”has shown by sequencing of 12 mutants that the mutation rate wasactually about 2 mutations in the pir116 gene. The pir116 gene wasamplified as an EcoRI-SstI fragment with oligonucleotides C8832(5-CTTAACGGCTGACATGGGAATTC-3′) (SEQ ID NO: 23) and C8833(5′-CGATGGGCGAGCTCCACCG-3′) (SEQ ID NO: 24). After digestion with EcoRIand SstI, the mutagenized fragment containing pir116 was cloned intopXL3830 in place of the “wild-type” pir116 gene.

Plasmids carrying mutagenized pir116 (“pir116*”) were transformed intoE. coli strain XAC-1, the parent of pCOR host XAC1pir116. Transformantswere screened for increased fluorescence under UV light and compared toXAC1 (pXL3830) and XAC1 (pXL3795) controls. A duplicate plate was notexposed to UV to minimize secondary mutations. A representativescreening plate under UV light is shown in FIG. 25.

The following flow chart summarizes the results of the screeningexperiment.

The evaluation of the three selected mutants is summarized in FIG. 26.Each mutant showed an increase in copy number as compared to the pir116plasmid. In the case of mutants 114C and 100B, the plasmid wasessentially in monomeric form. This could be an advantage as compared toa pir116pir42 plasmid, which has an increased copy number and a highmultimer content, like mutant 201C.

The pir116* gene of each mutant was sequenced. Each clone contains asingle non-isocodant mutation in pir116 ORF. All three of the mutationsaffect the C terminus of the pi protein, which is involved in DNAbinding. None of these mutations have been described before.

Once detected by screening in a plasmid system, these mutations wereevaluated in a production system, that is, where the pir116* gene isintroduced into the genome of an E. coli pCOR host strain. The strategyfor this evaluation is summarized in FIG. 27.

For this evaluation, plasmid pXL3179 plasmid was transformed into eachof the three E. coli strains bearing the mutations identified in FIG.26, and assessed for plasmid copy number and topology. The results ofthese experiments are presented in FIG. 28. It was observed that plasmidcopy number was significantly increased relative to XAC1pir116 only for201C mutant.

EXAMPLE 14 Minicircle with M13 Gene III as a Tool for Integration byHomologous Recombination in E. coli

1. Suicide Vectors

Gene replacement by double homologous recombination in E. coli requiresthe use of a suicide vector. These vectors are constructed and producedin a host capable of replicating them and used subsequently forrecombination into the chromosome of a host unable to replicate them.

Bacteriophage M13 is a very useful genetic tool that can be used in repmutants (Metcalf, W., W. Jiang, et al., Gene 138:1-7 (1994)) or innon-suppressor strains of E. coli when M13 mp 8 through 11 are used(Blum, P. et al., J. Bacteriol., 171:538-46 (1989)). Certain limitationsin terms of construction, insert size, and instability are frequentlyencountered. Plasmids carrying the R6K gamma DNA replication origin arewell known suicide vectors (Miller, V. and J. Mekalanos, J. Bacteriol.,170:2575-83 (1988)), but they are not useful for modifying E. colistrains that express the pi protein, which permits such plasmids toreplicate.

A universal suicide vector was engineered with a novel counterselectable marker and used to construct E. coli strains wherein mutantsof the pir116 gene (pir116*) are inserted into a bacterial genome byhomologous recombination. The strategy presented here is demonstratedfor mutant 114C, but has also been used to produce strains bearing otherpir116* mutants.

2. Counter-Selectable Marker

Different markers can be used to select for bacteria having undergone asecond recombination event. This event leads to the loss of this markerand in some cases to gene replacement after recombination between thechromosome and a suicide vector. For instance, the SacB gene fromBacillus is lethal when bacteria expressing the gene are plated on amedium containing sucrose (Ried, J. L. and A. Collmer, Gene 57:239-46(1987)). As another example, the tetracycline resistance gene conferssensitivity to fusaric acid (Bochner, B. R., et al., J. Bacteriol.(1980)). The infection by the bacteriophage M13 confers the sensitivityto the detergent deoxycholate (Blum, P., et al., J. Bacteriol.171:538-46 (1989)).

Due to a lack of efficiency in some E. coli strains, a positiveselection method for double recombinants was developed. Gene III frombacteriophage M13 was evaluated as a counter-selectable marker. Thisgene encodes a minor virion component responsible for the infectivity ofthe particles. When overexpressed from the multicopy plasmid pBR322,gene III confers deoxycholate sensitivity on the cells due to insertionof the gene III protein into the membrane of the bacteria (Boeke, J. D.et al., Mol Gen Genet. 186:185-92 (1982)). No report indicated if thisgene could be used as an efficient counter-selectable marker whenpresent as a single copy in the genome of the E. coli. Therefore, wetested this hypothesis with a minicircle suicide vector.

3. Amplification by PCR of the Deleted Version of Gene III from M13

To reduce the size of the minicircle vector to be constructed, a deletedversion of gene III (gene III′) that is still able to confer sensitivityto deoxycholate (Boeke, J. D., P. Model, et al., Mol Gen Genet.186:185-92 (1982)) was chosen. It was amplified by PCR along with itsown promoter from M13 mp18 (Yanisch-Perron, C., J. Vieira, et al., Gene33:103-19 (1985)) as a BglII-XhoI fragment (see FIG. 29).

The oligonucleotides were as follows:

C19519: (SEQ ID NO: 25) 5′-GGCAGATCTTAAACCGATACAATTAAAGG-3′        BglIIC19520: (SEQ ID NO: 26) 5′-CCGCTCGAGTTACGATTGGCCTTGATATTCACAAAC-3       XhoI

The amplified fragment was cloned by T-A cloning into pGEMT-easy(Promega Corporation, Madison, Wis., USA) to generate pXL4230 (FIG. 29).The nucleotide sequence of the insert was found to agree with thatdescribed in GenBank under accession no. VB0018. pXL4230 conferssensitivity to deoxycholate when transformed in E. coli strain DH10B(Invitrogen), indicating that it functions as expected.

4. Minicircle-Based Suicide Vector

As it does not contain any origin of replication, a minicircle plasmidmay be used as a universal suicide vector. For this purpose, aselectable marker such as the kanamycin resistance gene must be added toselect for a first homologous recombination event. To counter-select forbacteria that have not undergone a second event of recombination, thegene III′ was added to the minicircle vector. The construction of theplasmid used to produce minicircle for recombination is shown in FIG.29.

The minicircle is generated from a plasmid, such as pXL4235, afterinduction of the bacteriophage lambda integrase, which recombinesbetween attP and attB on the plasmid (Darquet, A. M et al., Gene Ther4(12): 1341-9 (1997)). This recombinase is expressed under the controlof P_(BAD) in a arabinose-dependent manner in E. coli strain G6264,which is described in U.S. application Ser. No. 09/981,803. Theresulting minicircle contains attL, a TH (triple-helix) forming sequencefor purification, the selectable marker Tn903 kanamycin resistance gene,the counter-selectable marker gene III′ and the fragment of interest forhomologous recombination, cloned in the multi-cloning site of pXL4235(FIG. 29).

As an example, the constructs used to generate E. coli strainsexpressing a copy-up mutation of pir116 are described in FIG. 30. Thesestrains can be used to produce pCOR plasmids (Soubrier, F. et al., GeneTher 6:1482-1488 (1999)). Since there is no homology between pir and thebacterial genome, the pir116* sequences were inserted into the E. colichromosomal uidA gene, which encodes β-D-glucuronidase. This geneprovides sufficient sequence similarity with the E. coli genome forhomologous recombination to occur.

The protocol for the purification of minicircle and recombinationoccurred as follows. Plasmid pXL4256 (FIG. 30) was transformed in E.coli strain G6264 to generate G6656. Fifty ml of LB medium supplementedwith ampicillin (100 mg/i) were inoculated with 0.5 ml of an overnightculture of G6656 and incubated at 37° C., with shaking at 200 rpm untilthe optical density at 600 nm reached 0.7. Minicircle production wasinduced by the addition of 25011 of a sterile solution of 10% arabinoseto the medium. After 30 minutes at 37° C., 200 rpm, total plasmid DNAwas extracted using the Wizard Plus Midipreps DNA Purification system(Promega Corporation, Madison Wis., USA).

Six μg of the plasmid DNA preparation were loaded onto a 0.8% agarosepreparative gel. A supercoiled DNA ladder (Promega Corporation, MadisonWis., USA) was used as a molecular weight standard. Afterelectrophoresis overnight at 50V, the supercoiled minicircle construct(5.1 kb) was extracted and purified from the gel using an SV gelpurification kit (Promega Corporation, Madison Wis., USA).

5. Double Homologous Recombination with Minicircle 4256 (uidA::pir116*Minicircle Suicide Vector)

The recombination steps for constructing the strains and thecorresponding phenotypes are described in FIG. 31.

For the first recombination event (integration), 0.2, 1 and 5 μl ofpurified minicircle 4256 were electroporated in E. coli strain XAC1(Normanly, J et al., Proc Natl Acad Sci USA 83:6548-52 (1986)), which isthe parental strain for pCOR hosts. Kanamycin resistant colonies wereobtained on LB Agar supplemented with kanamycin (50 mg/l) afterovernight incubation at 37° C.

To evaluate the number of colonies potentially containing contaminantnon-recombined pXL4256, 50 KmR colonies were streaked in parallel on LBAgar supplemented with kanamycin or ampicillin. Only 4 colonies out of50 were resistant to kanamycin and ampicillin and were shown by plasmidrestriction analysis to contain non-recombined pXL4256. This indicatedthat 46 colonies out of 50 obtained by electroporation were actuallyminicircle 4256 integrants.

For the second recombination event (excision), all of the 46 KmRintegrants were isolated on freshly prepared LB Agar plates containing1.5% sodium deoxycholate (“Doc”; Sigma) and incubated at 37° C.overnight. Only a few deoxycholate-resistant (DocR) colonies (1 to 15)were obtained for each integrant, as shown in FIG. 32. This result wasconsistent with the selection of a relatively rare event, such as thesecond recombination event. 100 DOc^(R) colonies obtained from 15integrants were patched in parallel on LB Agar with 1.5% Doc and LB Agarplus kanamycin to screen for Doc^(R) and Km^(S) double recombinants. 86%of the screened colonies were Km^(S), indicating that they had lost thesuicide vector.

To screen for allele replacement, the chromosomal uidA locus wasamplified by PCR. If allele replacement has occurred, the expected PCRfragment size is 1.3 kb. The fragment size corresponding to wild-typeuidA locus, that is, without an integrated pir116* mutation, is 0.85 kb.The results presented in FIG. 33-panel A indicate that allelereplacement has occurred in 30% of the double recombinants. This wasconfirmed by phenotypic analysis because these clones are also UidA-(beta glucuronidase-) and give white colonies on LB agar supplementedwith Xgluc.

The integrity of the bacterial genome in the region close to the site ofhomologous recombination was checked by PCR on two independentrecombinants. The first primer (seq6113 or seq6115) was based on thesequence of the pir gene and the second (seq6112 or seq6116) had asequence based on a sequence close to, but outside of, the homologyregion (immediately 5′ or 3′ of uidA). XAC1 DNA was used as a negativecontrol, whereas XAC1pir116 or TEX1 (Soubrier, F. et al., Gene Ther6:1482-1488 (1999)) were used as positive controls.

The oligonucleotides used as PCR primers were the following:

Seq11088: 5′-GAGATCGCTGATGGTATCGG-3′ (SEQ ID NO: 27) Seq11089:5′-TCTACACCACGCCGAACACC-3′ (SEQ ID NO: 28) Seq6112:5′-GACCAGTATTATTATCTTAATGAG-3′ (SEQ ID NO: 29) Seq6113:5′-GTATTTAATGAAACCGTACCTCCC-3′ (SEQ ID NO: 30) Seq6115:5′-CTCTTTTAATTGTCGATAAGCAAG-3′ (SEQ ID NO: 31) Seq6116:5′-GCGACGTCACCGAGGCTGTAGCCG-3′ (SEQ ID NO: 32)

The expected size for the PCR product is 0.83 kb using primers seq6112and seq6113, and 0.88 kb when using primers seq6114 and seq6115. Resultsare presented in FIG. 33-panel B. The two double recombinants obtainedwith the minicircle suicide vector showed the expected PCR profile. Thisdemonstrates that double homologous recombination can be easily achievedin E. coli using minicircle plasmids as suicide vector and the M13 geneIII′ as a counterselectable marker. This gene replacement technique canbe directly universally carried out in any micro-organisms geneticbackground.

BIBLIOGRAPHY

-   Alting-Mees, M. A., J. A. Sorge, and J. M. Short. 1992. Methods    Enzymol. 216:483-495-   Blum, P., D. Holzschu, H. S. Kwan, D. Riggs, and S. Artz. 1989. J.    Bacteriol. 171:538-546.-   Brosius, J. 1989. Methods Enzymol. 216:469-483.-   Chambers, S. P., S. E. Prior, D. A. Barstow, and N. P. Minton. 1988.    Gene 68:139-149.-   Chung, C. T., and R. H. Miller. 1988. Nucleic Acids Res. 16:3580.-   Colloms, S. D., P. Sykora, G. Szatmari, and D. J. Sherrat. 1990 J.    Bacteriol. 172:6973-6980.-   Datta, N., and P. Kontomichalou. 1965. Nature 208:239-241.-   Dickely, F., D. Nilsson, E. B. Hansen, and E. Johansen. 1995. Mol.    Microbiol. 15:839-847.-   Filutowicz, M., S. Dellis, I. Levchenko, M. Urh, F. Wu, and D.    York. 1994. Prog. in Nucleic Acid Res. and Mol. Biol. 48:239-273.-   Gibson, T. J. 1984. Ph.D Thesis. University of Cambridge.-   Greener, A., M. Filutowicz, M. McEachem, and D. Helsinki. 1990. Mol.    Gen. Genet. 224:24-32.-   Herrero, M., V. de Lorenzo, and K. N. Timmis. 1990. J. Bacteriol.    172:6557-6567.-   Hodgson, C. P. 1995. Bio/Technology 13:222-225.-   Inuzuka, M., and Y. Wada. 1985. EMBO J. 4:2301-2307.-   Jaye, M. et al., (1986) Science 233:541-5-   Kleina, L. G., J. M. Masson, J. Normanly, J. Abelson, and J. H.    Miller. 1990. J. Mol. Biol. 213:705-717.-   Kowalczykowski, S. C., and A. K. Eggleston. 1994. Annu. Rev.    Biochem. 63:9991-10043.-   Leung, D. W., E. Chen, G. Cachianes, and D. V. Goeddel. 1985. DNA    4:351-355.-   Maniatis, T., E. F. Fritsch, and J. Sambrook. 1989. Cold Spring    Harbor Laboratory, Cold Spring Harbor, N.Y.-   Meinnel, T., E. Schmitt, Y. Mechulam, and S. Blanquet. 1992. J.    Bacteriol. 174:2323-2331.-   Mertens, N., E. Remant and W. Fiers. (1995) Bio/Technology    13:175-179-   Messing, J., and J. Vieira. 1982. Gene 19: 269-276.-   Metcalf, W. W., W. Jiang, and B. L. Wanner. 1994. Gene 138:1-7.-   Miller, V. L., and J. J. Mekalanos. 1988. J. Bacteriol.    170:2575-2583.-   Normanly, J., J. M. Masson, L. G. Kleina, J. Abelson, and J. H.    Miller. 1986. Proc. Natl. Acad. Sci. USA 83:6548-6552.-   Normanly, J., L. G. Kleina, J. M. Masson, J. Abelson, and J. H.    Miller. 1990. J. Mol. Biol. 213:719-726.-   Roca, J. 1995. TIBS 20:156-160.-   Saiki, R. K., S. Scharf, F. Faloona, K. B. Mullis, G. T. Horn, H. A.    Erlich, and N. Arnheim. 1985. Science 230:1350-1354.-   Sanger, F., S, Nicklen, and A. R. Coulson. 1977. Proc. Natl. Acad.    Sci. USA 74:5463-5467.-   Sawadogo, M., and M. W. Van Dyke. 1991. Nucleic Acids Res. 19:674.-   Scott, J. R. 1984. Microbiol. Rev. 48:1-23.-   Simoes, D. A., M. Dal Jensen, E. Dreveton, M. O. Loret, S.    Blanchin-Roland, J. L. Uribelarrea, and J. M. Masson. 1991. Ann.    N.Y. Acad. Sci. 646:254-258.-   Simon, R., U. Priefer, and A. Pühler. 1983. Bio/Technology    1:784-791.-   Sinha, N. D., J. Biernat, J. McManus, and H. Koster. 1984. Nucleic    Acids Res. 12:4539-4557.-   Stirling, C. J. G. Stewart, and D. J. Sherrat. 1988. Mol. Gen.    Genet. 214:80-84.-   Stirling, C. J., S. D. Colloms, J. F. Collins, G. Szatmari,    and D. J. Sherrat. 1989. EMBO J. 8:1623-1627.-   Studier, F. W., A. H. Rosenberg., J. J. Dunn and J. W. Dubendorff    (1990). Methods Enzymol 185:60-89.-   Summers, D. K., and D. J. Sherrat. 1984. Cell 36:1097-1103.-   Takahashi, K., Y. Sawasaki, J. Hata, K. Mukai and T. Goto. (1990) In    Vitro Cell Dev. Biol. 26:265-74.-   Vieira, J., and J. Messing. 1982. Gene 19:259-268.-   Wiechelman, K., R. Braun, and J. Fitzpatrick. 1988. Anal. Biochem.    175:231-237.-   Yanisch-Perron, C. Vieira and J. Messing (1985) Gene 33:103-119 13.

1-49. (canceled)
 50. Plasmid pXL3179.
 51. A composition comprising theplasmid of claim 50 and a vehicle.
 52. A pharmaceutical compositioncomprising the plasmid of claim 50 and a pharmaceutically acceptablevehicle.
 53. A recombinant host cell comprising the plasmid of claim 50.54. The recombinant host cell of claim 53, wherein the host cellexpresses a n initiator protein.
 55. The recombinant host cell of claim54, wherein the host cell is Escherichia coli.
 56. The recombinant hostcell of claim 54, wherein the host cell is TEX1, as deposited underCollection Nationale de Cultures de Microorganismes accession numberCNCM 1-3569.
 57. A method of producing plasmid pXL3179, comprising a)culturing a recombinant host cell according to claim 53 under conditionspermitting replication of the plasmid, and b) isolating the plasmidproduced by the host cell.
 58. A plasmid comprising the nucleotidesequence set forth in SEQ ID NO:
 40. 59. A composition comprising theplasmid of claim 58 and a vehicle.
 60. A pharmaceutical compositioncomprising the plasmid of claim 58 and a pharmaceutically acceptablevehicle.
 61. A recombinant host cell comprising the plasmid of claim 58.62. The recombinant host cell of claim 61, wherein the host cellexpresses a π initiator protein.
 63. The recombinant host cell of claim62, wherein the host cell is Escherichia coli.
 64. The recombinant hostcell of claim 62, wherein the host cell is TEX1, as deposited underCollection Nationale de Cultures de Microorganismes accession numberCNCM I-3569.
 65. A method of producing a plasmid comprising thenucleotide sequence set forth in SEQ ID NO: 40, comprising a) culturinga recombinant host cell according to claim 61 under conditionspermitting replication of the plasmid, and b) isolating the plasmidproduced by the host cell.
 66. A plasmid consisting of the nucleotidesequence set forth in SEQ ID NO:
 40. 67. A composition comprising theplasmid of claim 66 and a vehicle.
 68. A pharmaceutical compositioncomprising the plasmid of claim 66 and a pharmaceutically acceptablevehicle.
 69. A recombinant host cell comprising the plasmid of claim 66.70. The recombinant host cell of claim 69, wherein the host cellexpresses a T initiator protein.
 71. The recombinant host cell of claim70, wherein the host cell is Escherichia coli.
 72. The recombinant hostcell of claim 70, wherein the host cell is TEX1, as deposited underCollection Nationale de Cultures de Microorganismes accession numberCNCM 1-3569.
 73. A method of producing a plasmid consisting of thenucleotide sequence set forth in SEQ ID NO: 40, comprising a) culturinga recombinant host cell according to claim 69 under conditionspermitting replication of the plasmid, and b) isolating the plasmidproduced by the host cell.